Biochimie 82 (2000) 269−288
© 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
S0300908400002017/FLA
Molecular genetics of regulated secretion in Paramecium
Laurence Vayssié, Fériel Skouri*, Linda Sperling, Jean Cohen**
Centre de Génétique Moléculaire, CNRS, avenue de la Terrasse, 91198 Gif-sur-Yvette, France
(Received 21 December 1999; accepted 10 February 2000)
Abstract — Paramecium is a unicell in which cellular processes are amenable to genetic dissection. Regulated secretion, which
designates a secretory pathway where secretory products are first stored in intracellular granules and then released by exocytotic
membrane fusion upon external trigger, is an important function in Paramecium, involved in defensive response through the release
of organelles called trichocysts. In this review, we focus on recent advances in the molecular genetics of two major aspects of the
regulated pathway in Paramecium, the biogenesis of the secretory organelles and their exocytosis. © 2000 Société française de
biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS
Paramecium / exocytosis / membrane fusion / trichocyst / secretory granule / multigene family
1. Introduction
The fascination Paramecium exercised over the 19th
century microscopists may have resulted from the animallike swimming behavior of the organism in response to
stimuli such as food, other paramecia or danger. Paramecium retreats from toxic substances such as acids, from
electric shock or from certain predators by swimming
backwards, leaving behind a trail of insoluble needleshaped objects first described as resembling hairs, hence
the name ‘trichocyst’ (figure 1) [1]. These trichocysts are
secretory products which result from a pathway of synthesis, storage and stimulus-dependent release that, at
least on a phenomenological level, is analogous to the
regulated secretory pathway that allows specialized metazoan cells to deliver secretory products (hormones, neuropeptides, digestive enzymes, histamine, etc.) to the
extracellular space in response to physiological stimuli
(for reviews see [2, 3]).
In this review, we shall describe work carried out over
the past decade on regulated secretion in Paramecium. It
has been an exciting period for Paramecium research as
* Present address: Laboratoire de Minéralogie Cristallographie,
UMR 7590 CNRS-Paris VI-Paris VII, 4, place Jussieu, 75252
Paris cedex 05, France.
* Correspondence and reprints: [email protected]
Abbreviations: AED, aminoethyldextran; DCV, dense core
vesicle; ER, endoplasmic reticulum; GRL, granule lattice
protein; IG, immature granule; kDa, kilo Dalton; ND, non
discharge; NSF, N-ethylmaleimide-sensitive factor; SDS-PAGE,
sodium dodecyl sulfate polyacrylamide gel electrophoresis;
SERCA, sarco(endo)plasmic reticulum-like calcium ATPase;
SNAP, soluble NSF attachment protein; SNARE, SNAP
receptor; TGN, trans Golgi network; TMP, trichocyst matrix
protein.
Figure 1. Trichocysts are secretory granules located at the cell
cortex. A. Phase contrast image of a living Paramecium showing
the cortical localization of the secretory granules (arrows). B.
Phase contract image of the intracellular, condensed trichocysts
that have spilled out of a lysed cell. C. Phase contrast image of
secreted trichocyst contents. The bars all represent 10 µm.
molecular genetics has developed considerably since the
last reviews on the topic ([4]; see also [5, 6]). While
‘classic’ biochemical, cytological, physiological and genetic analyses of regulated secretion have progressed, it
has in addition become possible to clone genes by
functional complementation and to inactivate genes by
homology-dependent gene silencing (for review see [7]).
Over the same period, decisive progress in studies of
vesicle trafficking, thanks to molecular genetics in yeast
and biochemistry in mammalian cells, has allowed identification of the basic machinery for membrane fusion [8,
9] and has brought considerable insight into the constitutive secretory pathway found in all eukaryotic cells,
270
whereby secretory products are packaged in vesicles,
transported to the plasma membrane and continually
released by vesicle exocytosis. The embellishments necessary for regulated secretion, involving the elaboration of
granules for intracellular storage of the secretory products
and their regulated exocytosis in response to extracellular
stimulation, are less well understood. Paramecium could
provide a powerful genetic system for the identification
and functional analysis of the additional (or different)
components required to build a regulated secretory pathway.
A variety of unicellular eukaryotes, covering considerably greater evolutionary distances than the whole of
metazoan evolution, have some form of regulated secretion. For example, Entamoeba histolytica, the parasitic
agent of amoebiosis, synthesizes electron-dense cytoplasmic granules which release collagenase and pore-forming
peptides [10, 11]; secretion of these granules is implicated
in degradation of host tissues.
It is however among the alveolates that the most
elaborate regulated secretory systems have been characterized. Part of the eukaryotic crown, the monophyletic
alveolata regroups ciliates, dinoflagellates and apicomplexan parasites according to recent molecular phylogenies [12, 13]. The common morphological trait of the
alveolata is the presence of a continuous membrane
system (‘alveolar sac’) underlying the plasma membrane
which, in Paramecium, has been shown to constitute a
vast calcium storage compartment much like the sarcoplasmic reticulum of muscle cells [14, 15]. Organisms
belonging to all three groups produce secretory granules
that are stored at the cortex, in close apposition to both the
plasma membrane and the alveolar membrane, a location
highly favorable for rapid exocytotic release. It is moreover striking that, at least in the apicomplexan Eimeria
(figure 2) [16], the secretory granules are inserted at
pre-formed cortical exocytotic sites which appear quite
similar to those found in ciliates judging by electron
microscope freeze fracture images. In the apicomplexan
parasites, which include the agents of widespread and
deadly diseases such as malaria, exocytosis of apical
granules is involved in host cell invasion [17]. In the free
living ciliates, exocytosis of cortical granules seems to be
involved in predator-prey interactions or secretion of
protective capsules.
Two lines of evidence speak for conservation ‘from
protists to neurons’ of the basic molecular machinery for
membrane trafficking. First, genome projects in apicomplexan parasites, in particular Plasmodium falciparum,
have already allowed the conceptual identification of two
key proteins, N-ethylmaleimide-sensitive factor (NSF)
and the t-SNARE syntaxin (table I). NSF as well as some
soluble NSF attachment proteins (SNAPs) have also been
identified in the genomes of the more primitive kinetoplastid parasites Leishmania major and Trypanosoma
brucei. Second, a recent study in the apicomplexan
Vayssié et al.
Figure 2. Exocytotic sites in the plasma membrane of Paramecium and the apicomplexan parasite Emieria. Freeze-fracture
electron microscopic images of an exocytotic site of Paramecium tetraurelia (courtesy of Claude Grandchamp) (A) and
Emeiria nieschulzi (B) (reprinted from [16] with permission)
showing typical arrays of intramembranous particles, the ring (ri)
and the central rosette (ro). Bar, 200 nm.
Toxoplasma gondii provides experimental evidence that
the NSF/SNAP/SNARE/Rab machinery is involved in
constitutive secretion in this organism and that the protozoan proteins can interact functionally with their mammalian homologues in permeabilized parasites [18]. Conservation of the molecular machinery for trafficking is not
surprising, given that it is now recognized that even the
most primitive eukaryotes have an endoplasmic reticulum
and Golgi apparatus [19].
Although the basic machinery is undoubtedly conserved, it is not yet clear whether the examples of
regulated secretion found in protists are evidence that the
pathway arose once in evolution, before the separation of
multicellular from unicellular organisms, and whether the
mechanisms, in their design and in their molecular details,
are conserved. Thus the very particularities that make
Paramecium an attractive experimental system, i.e., the
cortical localization of the granules and their elaborate
architecture, could either represent variations on a com-
Table I. Conservation of key membrane fusion proteins. Amino
acid identity for syntaxin (below the diagonal) and NSF (above
the diagonal) of human, yeast and Plasmodium falciparum
homologues. The latter are putatively identified on the basis of
sequence homology alone. The sequences were retrieved from
GenBank and compared using the BestFit program (Wisconsin
GCG package). Accession numbers: human syntaxin, U12918;
yeast SS02 syntaxin-like protein, P39926; P. falciparum putative
syntaxin, AAC71885; human NSF, P46459; yeast SEC18,
P18759; P. falciparum putative NSF, CAB10575.
Syntaxin\NSF
Human
Yeast
Protozoa
Human (%)
30
23
Yeast (%)
Protozoa (%)
44
51
52
23
Molecular genetics of regulated secretion in Paramecium
mon theme or a unicell’s innovative response to the
specific pressures for survival in a highly changing environment. Genetic dissection of the pathway in Paramecium could help answer this question, and is greatly
facilitated by the fact that trichocyst secretion is not an
essential function, at least under laboratory growth conditions, so that secretory mutants are perfectly viable.
In the first part of the review, we consider the problem
of granule biogenesis. Over the past 10 years, the secretory proteins that form the insoluble matrix of the trichocyst and their genes have been studied, in wild type
and secretory mutant cells. Pulse-chase experiments in
wild type cells and mutants have underscored the importance of protein processing in controlling biogenesis and
shown that defects in trafficking early in the pathway can
compromise granule formation. Molecular cloning revealed that a large multigene family encodes the secretory
proteins, and creation of mutant phenotypes by targeted
gene silencing indicates that the heterogeneity assured by
the multigene family is of functional significance.
In the second part of the review the final step of the
pathway, stimulus-dependent exocytosis, is considered.
The genetic dissection of this process is presented in
relation to associated physiological events such as calcium
movements. Molecular study of the mutants has begun
through complementation cloning and inactivation by
gene silencing.
2. Trichocyst biogenesis
2.1. Using the secretory pathway to build a molecular
spring
Paramecium trichocysts are secretory vesicles with at
least two unusual properties. First of all, they have a
highly constrained shape. As shown in figure 1B, each
trichocyst consists of a spindle-shaped body bearing at its
wide end a tip often compared to an inverted golf tee. The
tip is required for insertion of the trichocysts at preformed cortical exocytotic sites. Secondly, this object is
metastable. After exocytotic membrane fusion, contact
with the H2O and calcium ions in the external medium
leads to an extremely rapid (< 50 ms) and irreversible
expansion of the trichocyst contents, to yield a second,
needle-shaped form which remains insoluble (figure 1C).
Understanding how the Paramecium builds a trichocyst
thus means being able to account both for the spindleshape and the fact that in building up that shape, energy
has been trapped for later use. As trichocysts are a few
microns in size, their volume is about 1000 times that of
the dense core vesicles (DCVs) found in mammalian cells.
We have suggested that the trichocyst contents are designed as a molecular spring in order to facilitate the rapid
emptying of these huge vesicles upon exocytosis [20].
271
Figure 3. Trichocyst biogenesis. The schema represents the
different stages of trichocyst biogenesis, as visualized by electron microscopy. RE, endoplasmic reticulum. The solid gray
shading indicates amorphous vesicle contents, most likely consisting of uncleaved precursor molecules. Cross-hatching in
developing trichocysts indicates crystalline material, corresponding to the mature polypeptides.
A third property, which should help account for the first
two, is shared by many of the extrusomes found in protists
[21]. The vesicle contents have crystalline organization
[20, 22]. Explaining trichocyst shape thus consists in
identifying the factors that allow the secretory proteins to
crystallize in a controlled way within a membrane bound
compartment.
Before proceeding, it is important to realize that all of
these properties, even if they take on baroque proportions
in Paramecium and other protists, can be found in
mammalian DCVs. Although DCVs are usually unconstrained in shape, Wiebald Palade bodies found in endothelial cells are rod shaped, and their constrained shape is
conferred by the protein contents of the vesicles, as shown
by transfecting von Willebrand factor propolypeptide into
either pituitary or insulinoma cell lines [23]. Second,
secretory products are always condensed in DCVs so as to
be osmotically inert, and they de-condense as they disperse after exocytosis. Finally, the proteins stored in
DCVs can be present in crystalline form, insulin providing
a prime example [24].
The protein content of a trichocyst is a true protein
crystal, displaying periodicities in all three dimensions at
a resolution of about 50 Å according to X-ray diffraction
experiments [20]. However, this intracellular crystal is
composed, not of a single protein species but of a mixture
of small, acidic polypeptides which can be resolved into at
least 30 spots by two-dimensional gel electrophoresis [25,
26]. Moreover, all of this heterogeneity turns out to be at
the level of protein primary structure: the only post-
272
translational modification of these polypeptides known is
extensive proteolytic processing.
The preparation of polyclonal antibodies directed
against these polypeptides (trichocyst matrix proteins,
TMPs) and Western blot experiments with whole cell
extracts gave the first indication that the small polypeptides of the crystalline trichocyst matrix are the products
of proteolytic maturation of higher molecular mass precursor molecules [27]. Moreover, the precursor/product
ratio is perturbed in secretory mutants with aberrantly
shaped trichocysts, suggesting an important role for protein processing. Finally, when cells are fractionated using
a non-ionic detergent, the 40–45 kDa precursor molecules
are found only in the soluble fraction, while the
15–20 kDa polypeptides are insoluble, as expected since
they compose the crystalline matrix of the mature trichocysts.
Electron microscope immunolocalization experiments
with these antibodies provided direct evidence that a
classical secretory pathway is involved in trichocyst
biogenesis. Garreau de Loubresse [28] triggered massive
trichocyst exocytosis of a population of cells, then followed the wave of biosynthetic activity as the cells
reconstituted their stock of trichocysts. In this way, it was
possible to follow transport of the polypeptides recognized by the antibody from the endoplasmic reticulum
through the Golgi apparatus and into amorphous postGolgi vesicles. As these vesicles grew in size to about
1 µm (presumably by homotypic fusion events), crystallization began, probably at a point near the membrane.
Electron-dense tip material became visible at one end of
the vesicle as the final size of ≈ 2–3 µm was attained, and
the tip assembled completely only once the body matrix
had finished crystallizing. The schema for trichocyst
biogenesis that emerges from this study and the earlier
work of Estève [29] describing the organization of the
Golgi apparatus in Paramecium, is presented in figure 3.
We can now postulate that two kinds of genes are
required to make a functional trichocyst: 1) genes encoding the TMPs themselves; and 2) genes required for their
transport, sorting, and proteolytic maturation as well as
genes involved in regulation of the ionic composition of
the vesicles. Molecular characterization of the former,
which were cloned using a biochemical approach, is
described below in Section 2.3. The latter are not yet
cloned, however, we imagine that the many mutants that
affect trichocyst biogenesis arise from loss of function of
these genes. Biochemical and morphological studies of
mutants, presented in Section 2.2, support this hypothesis
and provide some insights into trafficking and protein
processing in Paramecium. Table II presents the genotypes and phenotypes of secretory mutants that have been
characterized so far in P. tetraurelia, the species most used
for genetic studies, in P. caudatum and in another ciliate
amenable to genetic analysis, Tetrahymena thermophila.
Vayssié et al.
2.2. Functions of protein processing
In order to study TMP processing in wild type and
mutant cells, we developed a pulse-chase protocol that
involves feeding Paramecium with metabolically labeled
bacteria for 10 min and chasing with cold bacteria [31]
(although axenic defined media have been developed for
Paramecium, this organism is usually cultivated using
monoxenic medium consisting of a grass infusion in
which bacteria have been grown, supplemented with a
sterol precursor [30]). Since digestive vacuoles are formed
roughly every minute and remain in the cell for about
20 min, the time resolution proved to be quite adequate for
studying processes with a half-life of 20 min or more.
Pulse-chase experiments using wild type cells (figure 4)
confirmed the precursor-product relationship between the
family of 40–45 kDa polypeptides seen on immunoblots
of whole cell extracts and the family of 15–20 kDa
polypeptides of the mature trichocyst matrix. The halftime of conversion in wild type cells was about 20 min.
The conversion was reversibly blocked by the drug
monensin, which in mammalian cells blocks protein
transport in the Golgi appartus, suggesting that the processing occurs in a post-Golgi compartment.
Examination of secretory mutants confirmed the previously postulated [27, 32] morphogenetic role of protein
processing in this system (figure 4): mutations that perturb
processing yield aberrantly shaped non-functional trichocysts, and one mutation which abolishes processing
yields cells with no trichocysts at all.
2.2.1. Sorting by retention: studies of the trichless mutant
Only one mutant has been isolated in P. tetraurelia that
has no trichocysts, the mutant trichless [33]. trichless cells
are characterized by slow growth (1–2 divisions per day as
opposed to 4–5 in wild type) and the total absence of
anything resembling a trichocyst. At the ultrastructural
level, these cells contain many clear vesicles as well as
small post-Golgi vesicles containing TMPs. The latter are
indistinguishable from the earliest immature granules
found in wild type cells, except for the fact that they are
coated by what appears to be clathrin [28, 34]. Since only
precursor molecules are present in trichless cells, this
provides an additional argument that TMP proteolytic
processing is a post-Golgi event.
Pulse-chase experiments with trichless cells (figure 4)
show: 1) only precursors are present; 2) they disappear
from the cell with a half-life of 40 min; and 3) no
degradation products are detected [31]. Biochemical
analysis of the culture medium revealed that precursor
molecules are constitutively secreted by trichless cells;
precursor molecules are undetectable in medium of wild
type cultures. Immunogold electron microscopy provided
some indication that the TMPs also end up in lysosomes in
trichless cells [34]. These data thus support ‘sorting by
retention’ of TMPs: the TMPs must crystallize in order to
Molecular genetics of regulated secretion in Paramecium
273
Table II. Mutants of the secretory pathway in Paramecium tetraurelia, Paramecium caudatum and Tetrahymena thermophila.
References in which information about the mutants is available are indicated in superscript, except for the non-discharge mutants
which are described in much greater detail in table III. Column 1. The phenotypic classes have been defined for mutants obtained in
P. tetraurelia. All these classes except the last one correspond to mutants unable to perform trichocyst exocytosis. The class cigar is
represented by mutations in the gene screwy (sc) which yield abnormal cell shape as well as abnormal trichocyst shape (cigar-like
instead of carrot-like). Column 4. Some mutants have been designated by different names in the literature: tam1 is also ndA, tam33
is t33, and nd2 is ndB.
1
Phenotypic class
in P. tetraurelia
2
3
4
5
6
Part of the
trichocyst
pathway altered
Phenotype
Mutants in P. tetraurelia
Equivalent
mutants in P.
caudatum
Equivalent mutants in T.
thermophila
[trichless]
[football]
Biogenesis
Biogenesis
[stubby]
Biogenesis
[pointless]
Biogenesis
[tam] (=
Transport
trichocyst defect
and
amacronucleate
cells)
tl4, 6, 7, 8, 9, 13, 18, 19, 23, 28, 30
ftA6,1,9,2,13 6, 18, 23, 30, ftB6, 23,
,
t33
tam381, 6, 7, 9, 12, 13, 17, 19, 20,
23, 30,
rug1 (27°C)31, rug231
short, unattached stA6, 9, 13, 18, 23, stB6, 23,
trichocysts
rug1 (18°C)31
unattached
ptA1, 18, 19, 23, ptB23
trichocysts
without tips
no trichocysts
abortive,
unattached
trichocysts
normal,
tam61, 3, 6, 12, 19, 23, tam106, 19
unattached trich.
tam12, 6, 18, 23,
- motile1
tam81, 2, 3, 4, 6, 7, 19, 23,
1
- not motile
tam116, 19
- motility not
checked
[non discharge]
[cigar]
Exocytosis
Biogenesis
SB28110, 11, 14, 15, 22, 25, 26
15, 22
SB28510,
,
10
SB255 /SB7155, 22, 25, 26
tam96, tam5432
attached but not nd2, nd3, nd6, nd7, nd9, nd12, tnd124, 29,
execretable
trichocysts
nd16, nd17, nd18, nd19, nd20, tnd224, 29
nd21, nd126, nd146, nd169,
nd203,
cam-1, d113
attached and
misshaped
trichocysts, but
execretable
MN173,11, 15, 27 SB28310, 22
MN17511, 15, SB28210, 15,
UC12.315, SB2585, 10, 16, 21,
SB25110
sc1-ci23
[118]; 2[78]; 3[54]; 4[57]; 5[119]; 6[72]; 7[28]; 8[34]; 9[31]; 10[120]; 11[121]; 12[77]; 13[41]; 14[122]; 15[123]; 16[124]; 17[98]; 18[33];
[60]; 20[51]; 21[125]; 22[126]; 23[127]; 24[116]; 25[128]; 26[129]; 27[37]; 28[130]; 29[81]; 30[131]; 31O. Garnier et al., unpublished
data; 32F. Ruiz, unpublished data.
1
19
remain in the regulated pathway. In the absence of any
processing, the soluble precursors are either secreted
constitutively or delivered to lysosomes. ‘Sorting by
retention’, as opposed to ‘sorting for entry’, was originally
proposed by Arvan and his colleagues to explain insulin
granule biogenesis in pancreatic β-cells [3, 35, 36].
Sorting of newly synthesized lumenal proteins (in this
case proinsulin) from lysosomal hydrolases, previously
thought to occur upon exit of the TGN, in fact continues
in immature secretory granules (IGs), which are an important sorting compartment for biosynthetic membrane traffic. It is now widely accepted that in professional secretory
cells a significant proportion of the molecules destined for
lysozomes or for constitutive export enter IGs and leave
them either through receptor-mediated sorting (the case of
lysosomal hydrolases recognized by the mannose-6phosphate receptor) or by ‘constitutive-like’ secretion
(soluble C-peptide). Insoluble granule proteins (such as
274
Vayssié et al.
Figure 4. Proteolytic processing of secretory protein control trichocyst biogenesis. On the left, transmission electron microscope
images of thin sections of wild type, tam38 and trichless mutant cells. Top, longitudinal section through a mature trichocyst, docked
at the plasma membrane in a wild type cell. bmx, body matrix; tmx, tip matrix; ts, trichocyst sheath. Middle, a typical tam38 trichocyst
characterized by an oval crystalline core, a highly abortive type and dense amorphous material between the core and the vesicle
membrane. Bottom, section of a trichless cell showing clear (small arrows) and dense (large arrows) post-Golgi vesicles in the vicinity
of a Golgi complex (Gc). The dense vesicles contain material recognized by anti-trichocyst antisera. Bar, 0.5 µm. On the right,
corresponding pulse-chase experiments, carried out at 27 °C. Log phase cultures of the appropriate strain were fed with metabolically
labeled bacteria for 10 min, washed, and transferred to chase medium containing unlabeled bacteria. Aliquots of the culture were
removed at the indicated times, lysed and immunoprecipitated with an anti-trichocyst antiserum. The immunoprecipitated proteins
were separated by SDS-PAGE and visualized by autoradiography. Reproduced from [31] with permission.
insulin) remain in the IGs owing to their aggregative
properties and the absence of specific sorting signals.
Since clathrin is involved in post-Golgi trafficking, the
coated appearance of the IGs in trichless cells is consistent
with ‘sorting by retention’. It will however be important to
test this hypothesis by seeing whether lysosomal hydrolases co-localize with TMP precursors in the IGs.
2.2.2. Kinetic control of crystallization
Mutations at several loci (see table II) give abortive,
aberrantly shaped trichocysts that cannot attach to the
cortex for lack of a complete tip assembly. Such mutants
grow slowly (2–3 divisions per day). For three of them
(tam33, tam38 and stubbyA), pulse chase experiments
gave very similar results (figure 4). Precursors are com-
Molecular genetics of regulated secretion in Paramecium
275
pletely converted to products, however the conversion is
significantly delayed compared to wild type: t1/2 of 70 min
instead of 20 min.
For one of the mutants, tam38, pulse chase experiments
were carried out at different temperatures. Although the
t1/2 of conversion increased as temperature was lowered,
at each temperature the rate of conversion was the same in
mutant and wild type cells, indicating that the processing
enzymes are not affected in the mutant. Evidence for a
delay in transport of the TMPs to the post-Golgi compartment where processing occurs was obtained by ultrastructural investigation of the three different mutant cell lines.
Massive accumulation of non-clathrin coated vesicles of
the type involved in ER to Golgi transport was found, as
in yeast SEC mutants. Thus trafficking defects early in the
secretory pathway can perturb protein processing in a
post-Golgi compartment. The fact that temporal perturbation in TMP processing compromises the assembly of the
crystalline matrix argues that matrix assembly is kinetically controlled [31].
lecular mass of 34 kDa [38]. Two-dimensional gel electrophoresis revealed the heterogeneity of the polypeptides
composing the trichocyst matrix [25, 26], with over 30
major polypeptides and as many as 100 minor ones of
molecular mass 14–21 kDa and with acidic isoelectric
points.
Improvements in separation of the mature polypeptides, by heat solubilization of TMPs that are not
disulfide-bonded [39] and by introduction of a chemical
spacer to improve resolution of isoelectric focusing [40],
set the stage for N-terminal microsequencing (reviewed in
[41]). Microsequences obtained for distinct 2D gel spots
were different, providing a first indication that TMP
heterogeneity is situated at the level of primary structure
[42]. The first cloning experiments, using the information
in the microsequences, revealed the existence of a large
TMP multigene family organized in subfamilies of very
similar genes [43].
Three subfamilies were characterized [43, 44]. Each
subfamily codes for distinct precursor molecules sharing
about 25% amino acid identity. However, within each
subfamily, four to eight genes sharing > 85% nucleotide
identity encode nearly identical proteins. All of these
genes seem to be co-expressed [43] and co-regulated at
the transcriptional level (Galvani and Sperling, see Note
added in proof). If we consider that a subfamily consists of
eight co-expressed genes, each of which yields two mature
polypeptides, then each subfamily can generate a maximum of 16 spots on 2D gels. As many of the spots
probably superimpose, this figure is likely to be an
overestimate. The three characterized subfamilies can thus
account, at most, for 48 of the 100 or so spots and we can
roughly estimate that there must be six to 10 different
subfamilies encoding different precursor molecules. This
figure is consistent with the fact that 10 distinct microsequences were obtained in our lab by sequencing 2D gel
spots [42], and by no means represent systematic exploration of the 2D map. Four of the microsequences are
found in genes from one of the three characterized
subfamilies (see figure 5) suggesting the existence, at the
very minimum, of three other subfamilies.
2.2.3. Setting the trap
Discussion of the functions of protein processing would
not be complete without mentioning two other functions,
despite the absence of any direct experimental data in
Paramecium. First, as originally pointed out with respect
to trichocyst biogenesis by Adoutte et al. [27], protein
processing can trap energy and create a metastable structure. When the polypeptide chain of a stable folded
precursor molecule is cleaved, a new conformation space
is opened up to it, which may include a thermodynamically more stable conformation. The trap is set. The
difference in energy between the actual and the more
stable conformation can be used to do work when the trap
is sprung. We imagine that this is why the trichocyst (like
other extrusomes) is a metastable structure. The trichocyst
uses the trapped energy to shoot itself out of the cell, when
the transition to the thermodynamically more stable state
is triggered by calcium ions.
The fact that calcium ions trigger the conformational
change in mature TMPs suggests an additional related
function for protein processing: sneaking the mature
calcium-sensitive polypeptides past the endoplasmic
reticulum, the cell’s primary calcium reservoir, by hiding
them in the precursor proteins. Some experimental evidence for this function has been obtained for the GRLs
(granule lattice proteins) in Tetrahymena, by comparing
the accessibility to proteolysis of precursors and mature
GRLs assayed at different calcium concentrations [37].
2.3. A large family of co-expressed genes encodes the
trichocyst matrix proteins
The earliest biochemical analysis of the TMPs, by
one-dimensional SDS-PAGE, reported that extruded trichocysts consist of disulfide-bonded dimers with a mo-
2.3.1. Novel processing enzymes?
Complete nucleotide sequences for one gene from each
of the three subfamilies were determined [45]. Alignment
of the deduced amino acid sequences revealed a common
organization (figure 5): each precursor yields two of the
mature polypeptides that crystallize, consistent with the
biochemical evidence that a given disulfide-bonded heterodimer is produced from a single precursor [32]. Surprisingly, the cleavage sites do not resemble known
endopeptidase cleavage sites, raising the possibility that
novel enzymes are involved. Furthermore, the site between the pro sequence and the first mature polypeptide is
clearly different from the site preceding the second mature
polypeptide (figure 5), suggesting that TMP processing
276
Figure 5. TMP precursors yield two mature polypeptides. The
organization of the precursors is shown schematically in the top
part of the figure and consists of a signal sequence (pre), a pro
sequence and two mature polypeptides separated by a basic
region. The lower part of the figure shows the sequences of three
different precursor molecules at the sites of cleavage between the
pro sequence and the first mature polypeptide and between the
basic region and the second mature polypeptide. A short consensus is found at the first cleavage site (TG/G or TG/D) which does
not correspond to any known endopeptidase cleavage site. The
second site is different. Note that only the T1b second mature
polypeptide N-terminus has been experimentally determined
while the other two are simply presented according to the
sequence alignment. Thus the amino acids ‘SK’ in the other two
sequences could be the actual cleavage site, and might be
recognized by an endopeptidase belonging to the subtilisin
superfamily.
requires at least two enzymatic reactions. The situation is
similar for the homologous T. thermophila GRL precursors, encoded by genes which define a smaller multigene
family in that there are no subfamilies but ‘only’ six or
seven unique genes specifying distinct but related precursor molecules [37].
2.3.2. A unique protein fold
Analysis of the aligned polypeptide sequences [45]
revealed conserved heptad repeats (repetitions of seven
amino acids with apolar residues in the first and fourth
position), indicative of coiled-coil interactions between
α-helices. Such interactions stabilize two-stranded parallel
coiled coils found in fibrous α-proteins such as myosin.
They also can stabilize antiparallel interactions between
shorter helices, in globular proteins. TMPs appear to be
globular proteins, as indicated by their charged to apolar
amino acid ratio (< 0.75; the ratio is > 1 in fibrous
proteins). Moreover, the conserved heptad repeats are
relatively short although several of them cover the regions
corresponding to the mature polypeptides. We therefore
proposed (with the kind help of David A.D. Parry) that the
Vayssié et al.
major portion of each mature polypeptide consists of an
antiparallel bundle of α-helices, probably a four-helix
bundle, a motif found in many globular proteins (figure 6).
Only one of the three precursor sequences (T2c precursor) contains cysteine residues, consistent with the fact
that the original microsequence used to clone T2 subfamily genes was generated from a disulfide-bonded heterodimer [26]. The two other precursor sequences (T1b
and T4a precursors) contain no cysteines at all (except in
the T1b pro sequence), consistent with the fact that both
genes were cloned using microsequences of mature
polypeptides that belong to the minority class of heatsoluble monomers [46]. The positions of the two cysteine
residues in the T2c precursor, between the first and second
helices of the first mature polypeptide and between the
second and third helices of the second mature polypeptide,
fit very nicely with the proposal of a four-helix bundle
motif. We were able to propose a model for the pseudosymmetric arrangement of two four-helix bundles within a
precursor molecule (figure 6B, C). The most important
implication of the analysis, whether or not the details turn
out to be correct, is that all of the TMPs share the same
polypeptide fold, despite their different primary structures.
2.3.3. TMP multigene family: redundancy or necessity?
The existence of a large multigene family, encoding
secretory protein precursors with the same organization
and the same protein fold, raises the question of its
functional significance. It seems possible that all of the
mature TMPs, four-helix bundles with similar 3D structures, are interchangeable in the crystal lattice of the
trichocyst matrix. The function of the multigene family,
which would have accumulated neutral mutations consistent with the protein fold, might then be to assure
synthesis of large amounts of protein. Alternatively, the
chemical heterogeneity among the TMPs could be necessary to build up a functional crystalline edifice with a
constrained shape.
Biochemistry and genetics have so far provided strong
evidence in favor of the more attractive hypothesis that
Paramecium generated and maintains the TMP multigene
family because all of the genes are necessary to build a
functional secretory granule. We produced sequencespecific polyclonal antibodies by expression of synthetic
genes corresponding to different mature polypeptides [47]
and are currently using them to study trichocyst biogenesis. The results so far confirm and extend data published
by Hausmann et al. [48] and Shih and Nelson [32, 49].
These groups performed immunolocalization experiments
using monoclonal antibodies and reported that most of the
different mAbs, which recognize different subsets of
mature TMPs, decorate one or the other of two nonoverlapping concentric regions of the mature trichocyst
matrix, an inner core and an outer cortex. This pattern
suggests that the polypeptides are not interchangeable, but
assemble in a specific temporal order.
Molecular genetics of regulated secretion in Paramecium
277
Figure 6. TMP proposed protein fold. A. Schematic drawing of a ‘generic’ four α-helical antiparallel bundle, with a left-handed tilt
necessary to optimize coiled-coil packing of the helices. The chain connectivity has been arbitrarily drawn as right-handed. This is the
protein fold proposed for each of the mature polypeptides. B. Possible arrangement of two mature polypeptides in a precursor
molecule, showing the disulfide bond that could be formed in a T2c precursor. The two bundles are facing each other, related by a
pseudo two-fold symmetry axis. C. Birds-eye view of the precursor molecule in B, showing that the connectivity is the same for each
bundle and for the molecule as a whole. Reproduced with modifications from [45] with permission.
A strong genetic argument that TMP genes are not
functionally redundant is provided by gene silencing
experiments. Briefly, homology-dependent gene silencing
is a phenomenon whereby the introduction of the coding
region of a gene into the Paramecium somatic nucleus at
high copy number leads to specific reduction in the
expression of all endogenous homologues of the gene. In
this way, we were able to specifically reduce the expression of either the T1 or the T4 subfamily. In both cases,
exocytosis-deficient phenotypes were obtained. The si-
lenced, exocytosis-deficient cells contained unattached,
aberrantly shaped trichocysts in their cytoplasm (figure 7).
Most significantly, the shapes of the trichocysts were
different depending upon which subfamily was silenced.
These experiments clearly demonstrate that the different TMP subfamilies are required for trichocyst biogenesis
and have different functions in that process. Since silencing reduces expression (to ≈ 25% of the level of expression of genes belonging to the other, non-silenced subfamilies [50]) but does not abolish it, we can also postulate
278
Vayssié et al.
released by fusion of the granule membrane with the
plasma membrane. The peculiarity of Paramecium is: 1)
that the secretory granules (trichocysts) are docked at the
cortex in close apposition to the plasma membrane; and 2)
that the docking sites are predetermined. After biogenesis
as free granules within the cytoplasm, trichocysts have to
be transported to their attachment site. As already shown
in Section 2, mutants can be obtained which cannot
undergo exocytosis, since trichocyst discharge is not an
essential function for cell life in laboratory conditions. In
addition to biogenesis mutants, transport and exocytosis
mutants can be obtained (table II).
3.1. Trichocyst docking
Figure 7. Immunofluorescent images of cells in which T1 or T4
genes have been silenced. A. T1 silenced cell underneath a wild
type cell. B. T4 silenced cells. The silenced cells present
undocked aberrantly shaped trichocysts. Reprinted from [50]
with permission
that the precise stoichiometry of the different TMPs is
important, suggesting the existence of subassembly pathways in trichocyst biogenesis. Existence of subassemblies
of TMPs was previously suggested when buoyant density
measurements indicated that the elementary unit cell of
the trichocyst crystal lattice (111 Å × 130 Å × 330 Å) can
accommodate ≈ 50 mature polypeptides [20].
2.4. Perspectives: trichocyst shape as a screen for
traffıcking mutants
The mendelian mutants we have studied the most
extensively, trichless [33] and tam38 [51], were isolated in
the 1970s by morphological observation. Given the question raised in the Introduction about the conservation of
molecules involved in membrane trafficking, it now seems
worthwhile to clone these and other biogenesis genes. It
would also be worthwhile to screen for new biogenesis
mutants, something that has not been done systematically
since the pioneering work of Pollack [33], for the system
is far from saturated. We can now hypothesize that many
of the genes that can be identified in this way should be
involved in intracellular transport. At least some of them
should be Paramecium homologues of NSF/SNAP/
SNARE/Rab machinery components [9]. We also anticipate that cloning biogenesis genes could reveal components specific to granule biogenesis, involved in postGolgi steps of the process. It will be particularly
interesting to see whether known processing enzymes are
involved in maturation of the precursors, or whether we
will be able to identify new enzymes.
3. Trichocyst exocytosis
Exocytosis refers to the ultimate step of the secretory
pathway where the contents of secretory granules are
Trichocysts are transported to the cell cortex through
saltatory movements [52], likely along microtubules [53].
The whole transport and attachment process is sensitive to
cytochalasin and this drug produces phenocopies of mutants with undocked trichocysts known as the tam mutants
[54]. Microfilaments of actin and microtubules may therefore both play an essential role in the transport and
recognition of the docking sites by trichocysts.
The predetermined cortical sites are characterized,
when observed by freeze-fracture electron microscopy, by
intramembranous particle arrays with a shape of parentheses, likely delineating the junction between the edges of
the underlying alveolar sacs and the plasma membrane.
When trichocysts reach the cortex, the parentheses transform into a ring, with the appearance of a central rosette of
particles (figure 8) [55–57], within 5 min after trichocyst
attachment [58]. At the same time, a fibrous material (the
‘plug’) present in the space between the alveoli is remodeled around the trichocyst tip and dense material organizes
to connect the trichocyst membrane to the plasma membrane, just underneath the rosette particles [59, 60]. The
trichocysts then remain anchored at the plasma membrane
as long as the cell is unstimulated, and are discharged
within milliseconds upon reception of an exocytotic
stimulus. The role of the docking structures is therefore
two-fold, allowing instantaneous membrane fusion in
response to stimulation, but also preventing erratic discharge when the cell is at rest.
3.2. Triggering exocytosis.
Like in all regulated secretory pathways, trichocyst
exocytosis is triggered by external stimuli. The close
apposition of the trichocyst membrane to the plasma
membrane, and anchoring through connecting material
and rosette, permits a very rapid response which is limited
to signal reception, signal transduction and membrane
fusion. Secretory stimuli include the fixative picric acid,
used to assay exocytotic performance of individual cells
(figure 9) [33], the vital polyamine aminoethyldextran
(AED [61, 62]) and lysozyme [63]. Trichocyst release is
Molecular genetics of regulated secretion in Paramecium
279
Figure 8. Wild-type and nd trichocyst exocytotic sites. The middle panel (C, D) presents a schematic view of the organization of a
trichocyst docking site in wild-type and nd mutants and is illustrated by electron microscope images of freeze fractures of plasma
membrane (A, B) and of thin sections (E, F) where the connecting material (cm) between the trichocyst membrane (tm) and the plasma
membrane (pm) and a rosette (ro) of intramembranous particles located at the center of a double ring (ri) of particles are represented.
am, alveolar membranes delineating the subplasmalemmal calcium stores; tt, trichocyst tip. A, C, E. Wild type docking site. B, D, F,
nd-type docking site in the cam1 mutant, displaying an nd phenotype at the non-permissive temperature. The rosette and the
connecting material are present in the wild-type site but not in the mutant. A, B, E, F. Reproduced from [71] with permission. Bars,
0.2 µm.
observed within a few milliseconds [64]. In natural
conditions, trichocyst discharge seems to have a defensive
function against attacks by predator ciliates such as
Dileptus [65] through a mechanism of escape [66, 67].
After membrane fusion, the release itself is thought to be
spontaneously performed by matrix expansion (release of
the ‘molecular spring’, see Section 2.2) when the interior
of the trichocyst comes into contact with the calcium-rich
external medium [68–70]. However, the mechanism by
which membrane fusion itself is induced is still unknown,
despite the fact that a prominent role of calcium has been
documented (see Section 3.6). Genetic dissection, which
has been underway for almost three decades, will perhaps
lead to deeper understanding of the exocytotic process.
3.3. The nd mutants identify genes necessary for
connecting material and rosette assembly
3.3.1. A panel of exocytosis mutants
The mutants called nd, for ‘non-discharge’ (table III),
display defects only in post-docking steps of the pathway,
namely signal reception, transduction, and exocytotic
280
Vayssié et al.
connecting material assembly and one can expect to find
calmodulin and/or calmodulin-binding proteins in these
structures.
3.3.3. ND gene products of different origins build the
connecting material
Figure 9. Evaluation of the exocytotic capacity of Paramecium
cells using the fixing secretagogue picric acid. The two upper
cells have an exocytosis deficient (exo-) phenotype. The lower
cell has a wild-type phenotype and is surrounded by a dense halo
of secreted trichocysts (exo+). Reprinted from [50] with permission.
membrane fusion, without alteration of prior events such
as biogenesis or transport. Altogether, 31 alleles defining
17 genes (including the calmodulin gene in which the
particular mutation cam-1 yields an nd phenotype at high
temperature [71]) have been obtained so far, either by
mutagenesis [33, 72, 73] or from wild type stocks [74, 75].
Given the number of genes for which we have only one
mutated allele (10 out of 17), we can anticipate that the
system is not saturated and that new mutageneses will
reveal new ND genes. Non-Mendelian nd mutants also
exist in which the phenotype is inherited maternally, for
example the strain d113 [75] or lines derived from crosses
between the mutant mtFE and the wild type but with wild
type genotype [76].
3.3.2. Abnormal exocytotic sites in nd mutants
Examination of the exocytotic sites of 17 nd mutants
(representing 13 genes), by freeze-fracture and transmission electron microscopy, revealed that all of them but one
(nd12-1) display severe defect or complete lack of the
rosette and connecting material assembly (table III) [57,
60, 71, 73, 77]. This high number of mutants presenting
such defects suggests that many proteins are components
of these structures or control their assembly. This is also
true for the only nd gene for which the function of the
product is known, the calmodulin gene. There should thus
be a calmodulin-sensitive step necessary for rosette and
Components of the connecting material and rosette
seem to come from products linked to three different
cellular compartments, the trichocyst, the plasma membrane and the cytosol. Indeed, it is possible to determine
the site of action of the mutations by rescue experiments
involving transfer of cytoplasm and trichocysts from cell
to cell by microinjection [73, 77, 78]. By this test, the gene
products ND3p and ND6p have been assigned to the
cortex, ND9p and ND16p to the cytosol, and ND2p,
ND7p, ND126p and ND169p to the trichocyst compartment. The site of action of the macronuclear mutation
d113 has been localized to the trichocyst [79]. All the nd
gene products therefore cooperate in the edification of a
single structure, whatever their compartment of origin.
A refinement of the determination of the site of action
is provided by experiments of conjugation rescue. Indeed,
a few hours before nuclear exchange, membranes from
conjugating cells fuse allowing contacts between the two
cytosols and continuity between the two plasma membranes. A rescue of the phenotype of the nd partner during
conjugation between an nd and a wild type occurs both in
‘cytosolic’ mutants (nd9-1) and in ‘cortical’ mutants
(nd6-1), as determined by microinjection. Careful observation can even allow distinctions to be drawn between
the modes of rescue: polarized from cell contacts in the
case of ‘cortical’ mutants and non-polarized in the case of
‘cytosolic’ mutants [77].
3.3.4. Interacting partners in the exocytosis site
Genetic interactions observed between genes suggest
direct protein-protein interactions between their products.
Negative interactions have been found between ND9,
ND16 and ND18 in the form of a more severe phenotype
in double mutants (exocytosis defect at all temperatures)
than in either of the single parental mutants (thermosensitive defect) [73]. Preliminary experiments also suggest
similar interactions between ND16 and ND203, ND203
and ND3, ND203 and ND17, and ND17 and ND9 (Nyberg
and Cohen, unpublished). The presence of so many
proteins, likely to interact with each other, in the connecting material and the rosette (or involved in their assembly)
is reminiscent of the exocytotic NSF/SNAP/SNARE/Rab
machinery found in other eukaryotic cells. Whether some
ND genes are homologs of these proteins is not known,
but the overall organization and mechanism of orchestrating membrane fusion are likely to be similar. In Paramecium, the exocytotic process is frozen in a prefusion state.
This may be part of a mode of fusion peculiar to this
unicell to ensure an instantaneous defensive function, or
represent a general but very transient step in other cell
types, which, by chance, is stable and visible in Parame-
Molecular genetics of regulated secretion in Paramecium
281
cium. Among the nd mutants with altered rosette and
connecting material, nd9-1 is the best-known [80]. Combined experiments of transfer of cytoplasm and trichocysts
by microinjection, temperature shifts of cultures, and
freeze-fracture analyses lead to the conclusions that the
gene product ND9p is a cytosolic factor that interacts with
both trichocyst and plasma membranes in a way sensitive
to the lipidic composition of the membranes. In addition,
the occurrence of interallelic interactions between nd9-1
and nd9-3 [73], suggests homopolymeric interactions
between ND9p gene products. Altogether, the data suggest
that ND9p is a component of the connecting material that
lies at the interface of this material with both membranes.
The network of genetic interactions between ND9 and
other ND genes confirms that ND9p should be a key
molecule in the edification of the exocytotic sites.
Table III. Phenotypic characteristics of the nd mutants of Paramecium tetraurelia and their equivalents in Paramecium caudatum and
in Tetrahymena thermophila. Features presented in columns 4–8 are from the nd mutants in non-permissive conditions, when they have
a thermosensitive phenotype. Column 1. Names of mutants are written according to the new genetic nomenclature rules [134]: for
example, nd9a becomes nd9-1, nd12 nd12-1 and cam1 cam-1; MAC, of maternal (macronuclear) inheritance; st, sterile line which
cannot be crossed; P.c.: Paramecium caudatum; T.t.:Tetrahymena thermophila. Column 2. – indicates no exocytosis; ε means that only
a few trichocysts (less than 10 per cell) can be discharged; +/- is for partial exocytosis of 50–300 trichocysts per cell; + indicates
complete exocytosis of a cloud of > 500 to 1000 trichocysts per cell. Column 3. The genes that have been cloned by functional
complementation are indicated. The cloning of cam-1 (calmodulin gene) was performed after calmodulin had been identified as the
rescuing factor for ciliary reversal abnormalities and after the mutant protein had been sequenced. The nd phenotype was discovered
in cam-1 cells only later, when the gene was already cloned. Preliminary experiments of rescue by DNA injection into the macronuclei
of a tnd2 mutant of P. caudatum have been reported [135]. Column 4. For all the strains indicated, the presence/absence of a rosette
of intramembranous particles at the exocytosis site has been checked by freeze-fracture electron microscopy. Column 5. The
presence/absence of connecting material between the trichocyst membrane and plasma membrane has been checked by transmission
electron microscopy for only a few strains. In these strains, the presence/absence of connecting material was correlated to the
presence/absence of a rosette. Column 6. Site of action of the mutation as determined by cytoplasm transfer rescue experiments. trich,
trichocyst; pm, plasma membrane/cortex; cyt, cytosol; not cyt, not cytosol, either plasma membrane or (most likely) trichocyst. In the
case of nd9-1 and nd9-3, high-speed supernatant of whole cell extracts as well as ammonium sulfate precipitates of this supernatant
are able to transiently rescue the mutants by microinjection into the cytoplasm (Cohen, unpublished). Column 7. Conjugation rescue
observed during mating and before nuclear exchange. Column 8. Application of a calcium ionophore can lead to exocytosis,
pseudo-exocytosis (trichocyst matrix stretching within the cell without membrane fusion), or no effect*.
1
2
3
4
Mutant
Exocytotic capacity at
Cloning
Rosette
yes10
yes
no27
Wild Type
nd2-11, 6, 7, 17, 22
nd2-228
nd3-17, 22
nd3-27, 22
nd3-37
nd3-46, 15, 22
nd3-56
nd3-627
nd6-16, 7, 12, 22
nd6-26, 15
nd6-326
nd7-15, 22
nd9-14, 6, 7, 12, 18, 22
nd9-26, 7
nd9-36, 7
nd12-17
nd16-17
nd16-26
nd16-326
nd17-17
nd18-16
nd19-126
nd20-116
18°C
27°C
35°C
+
–
–
–
–
+
–
–
–
–
ε
–
–
+
+
–
+
+
+
+
+
+
+
+
–
–
–
–
+
–
–
–
–
–
–
–
–
+
–
+
+
+/–
+
+
+
+
+
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
5
6
Conn. Mat. Site of action
of the
mutation
7
Conjug.
rescue
yes
8
Effect of Ca
ionophores
exocytosis
trich.2
no27
no6
pm6
yes10
no12, 18
no18
pm12, 18
yes12
yes21
no12, 18
no4, 5, 6, 12, 18
no27
no27
yes18
no27
no6
no18
no18
trich.12, 18
cyt.2, 5
no12
yes.5, 12
yes10
no27
no6
cyt.6
yes18
cyt.6
pseudoexo.13
282
Vayssié et al.
Table III. Continued.
1
2
3
4
Mutant
Exocytotic capacity at
Cloning
Rosette
26
nd21-1
nd126-16, 15
nd126-25, 6, 15
nd146-16, 15, 22
nd146-26, 15, 22
nd169-16, 15
nd203-16, 15, 22
cam-111
d113 (MAC)23
tnd1-ND11 P. c.24
tnd1-yt4s2 P. c.24
tnd1-16D317 P. c.24
tnd1-16D202 P. c.24
tnd2-yt3G9 P. c.24
tnd2-27aG3 P. c.24
tnd2-yt3S43 P. c.24
tnd2-C103 P. c.24
UC12.3 T. t.8, 14
MN175 T. t.8, 14
SB282 T. t.8, 14
SB258 T.t. (st.)8, 16, 19
SB251 T. t. (MAC)8
18°C
27°C
35°C
+
–
–
+/–
+/–
–
+/–
+
–
+
–
–
+/–
+/–
–
+/–
+
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
+/–
+/–
–
–
–
–
–
yes9, 20
5
Conn. Mat. Site of action
of the
mutation
no6
trich.6
no6
no6
no11
trich.6
24
yes
yes24
no24
no24
no24
–
6
no16
7
Conjug.
rescue
8
Effect of Ca
ionophores
no11
trich.3
not cyt.25
no25
cyt.25
pseudoexo25
none25
yes25
yes14
no14
exocytosis14
yes19
*All available references are noted in superscript. 1[118]; 2[78]; 3[79]; 4[57]; 5[80]; 6[73]; 7[72]; 8[120]; 9[132]; 10[93]; 11[71]; 12[77];
13
[95]; 14[123]; 15[74]; 16[124]; 17[33]; 18[60]; 19[125]; 20[133]; 21[92]; 22[127]; 23[75]; 24[116]; 25[81]; 26O. Garnier et al.,
unpublished data; 27M. Rossignol, unpublished data; 28F. Ruiz, unpublished data.
Interestingly, genetic interactions have also been found
in the double mutant tnd1-tnd2 of P. caudatum [81], but
with a different phenotype: trichocysts are mostly detached from the cortex, as in the tam mutants of P.
tetraurelia whereas single mutants are of the nd type
(table II). This could indicate that some molecules can
function both in trichocyst docking at the cortex and in
exocytotic membrane fusion but that the docking function
of the molecules is revealed only when two partners are
affected at the same time. This kind of interaction has not
been found yet in P. tetraurelia but could be linked to the
fact that one of the P. caudatum mutants (tnd1) displays a
normal rosette. Perhaps a similar situation will be found in
double mutants involving nd12-1, which also displays a
normal rosette at non-permissive temperature.
3.4. nd mutants and other genes?
Some of the nd mutants have been included as controls
in specific analyses on exocytosis-associated features
compiled in table IV (fate of calcium, of ATP, state of
phosphorylation, etc.). Such an approach has provided a
wealth of useful information, although it does not tell us
yet whether the defects harbored by the mutants are direct
or secondary effects of the altered gene products. Owing
to the new method of gene inactivation by homologydependent gene silencing [50], it will now be of interest to
inactivate genes suspected to have a role in exocytosis.
Among those worth studying, we can cite genes encoding
calcineurin (Russel and Hinrichsen, accession number
AF014922), small G-proteins [82] (some of which are
associated
with
trichocysts
[83]),
parafusin/
phosphoglucomutase [84, 85] (although, in this case, the
homologous protein in Tetrahymena has been shown by
gene disruption to have no essential role in exocytosis
[86]), SERCA-type calcium pump [87], guanylyl-cyclase
[88] and copine, a calcium-dependent lipid binding protein [89]. Any exocytosis defect induced by inactivation of
such genes will give further insights into the mechanism
of exocytosis since, with this approach, we start from
known functions and look for a phenotype.
Molecular genetics of regulated secretion in Paramecium
283
Table IV. The nd mutants of Paramecium tetraurelia as controls in the study of exocytosis. Column 2. Ca-uptake refers to a transient
calcium influx triggered by AED. Column 3. Ca-transients correspond to strong increase of intracellular calcium concentration at the
site of AED application, followed by a spill-over into deeper cell regions [115]. Column 4. Parafusin [140], a 63–65-kDa
phosphoprotein identified by Gilligan and Satir [136] as dephosphorylated upon stimulation of exocytosis, has also been called PP63
[141], PP65 [142] and phosphoglucomutase [143]. The dephosphorylation is very transient (5 s) and followed by rephosphorylation
[131]. This protein is also covalently modified with a glucose 1-phosphate and is dephosphoglycosylated during stimulation of
exocytosis [100]. Column 5. Parafusin has been shown by immunofluorescence to be associated with exocytosis sites and the
trichocyst membrane. Triggering exocytosis completely dissociates parafusin from these membranes, although the intracellular amount
remains stable. Column 6. Calcium-dependent phosphodiesterase activity able to dephosphoglycosylate parafusin. Column 7.
Calcium-dependent ATPase activity present at exocytosis sites, first described by in situ labelling of the wild type by Plattner et al.
[144]. Column 8. Calcium-dependent ATPase stimulated by calmodulin possibly associated with membranes. Column 9. ATP decay
refers to a transient (3-5 s) ATP consumption when cells are triggered for exocytosis. Column 10. Microinjection of calcium/
calmodulin/calcineurin, as well as of alkaline phosphatase, into the cytoplasm of Paramecium cells triggers massive exocytosis*.
1
Mutant
Wild type
nd6-1
nd7-1
nd9-1
nd12-1
2
3
4
5
6
7
8
9
10
Ca-uptake.
Catransients.
Parafusin
dephos.
Parafusin
removal
from tric.
Ca-dep
phosphodiesterase
Cortical
Ca-ATPase
CAMATPase
ATP decay
Exocytosis
induced by
phosphatase
injection
yes
yes1
yes1, 2
yes1, 2
no1, 2
yes
yes
no4
no4
no4, 5
yes
yes
yes
no8
yes
yes
yes10
yes
no6
no7
no8
no9
yes10
no11
yes3
*All available references are noted in superscript. 1[137]; 2[105]; 3[97]; 4[136]; 5[131]; 6[100]; 7[139]; 8[98]; 9[99]; 10[130]; 11[138].
3.5. From mutants to genes
In recent years, a sib-selection method was developed
in Ching Kung’s laboratory (Madison, Wisconsin, USA)
to clone Paramecium genes by functional complementation of mutants [90, 91], and adapted in our laboratory to
clone the ND7 gene [92]. Since then, in order to facilitate
systematic cloning projects, in particular of genes implicated in the secretory pathway, an indexed library of ca.
60 000 clones has been constructed [93] that already
permitted the very recent cloning of three new ND genes,
ND2, ND6 and ND9. The sequence of the gene ND7, as
well as preliminary data on the genes ND2 and ND9,
indicate that all three genes are novel ones, as they have
no equivalent in other species in the available data bases,
although functional domains can be recognized. Further
analysis of these genes and their products will increase our
understanding of the mechanisms developed by living
organisms to fuse membranes, complementary to the data
that will arise from the gene silencing approach.
3.6. Calcium movements during trichocyst exocytosis
Calcium has long been suspected to have a pivotal role
in signal reception, signal transduction and membrane
fusion in trichocyst exocytosis. Indeed, the introduction of
calcium into cells by ionophores [94, 95] or by microinjection [96, 97] induces exocytosis. In addition, correlations have been made between the exocytotic capacity and
the presence of calcium-related activities: a cortical cal-
cium ATPase [98], a calmodulin-dependent ATPase [99],
calmodulin [71] and a calcium dependent phosphodiesterase able to dephosphoglycosylate parafusin [100].
The possible sources of free calcium ions are two-fold:
entry from the external medium in which calcium concentration is in the millimolar range, compared to the submicromolar range in the cytosol [97, 101], or release from
the ‘cortical alveoli’ (subplasmalemmal calcium stores
[14]). The latter, whose calcium concentration is between
3 and 5 mM [15], shares properties with sarcoplasmic
reticulum in muscle cells, such as the sensitivity to
caffeine [102], the presence of a calsequestrin-like protein
[103] and the presence of a SERCA-type calcium ATPase
[87, 104].
3.6.1. Calcium influx…
The existence of a strong calcium influx was first
demonstrated to be associated with exocytosis upon AED
stimulation [105], within less than 80 ms [106]. Veratridine, another secretagogue in Paramecium [106], was also
shown to activate a somatic (non-ciliary) calcium channel
[107, 108]. The same calcium influx could indeed be
measured using veratridine or AED [109]. The discovery
of a calcium channel activated by hyperpolarization in the
membrane of Paramecium and sensitive to amiloride and
divalent cations [110] stimulated the examination of the
effects of these substances on exocytosis and associated
calcium influx. Erxleben and Plattner [111] found no
effect of these compounds on exocytosis. In contrast,
possibly due to methodological differences in stimulation,
284
Kerboeuf and Cohen [109] found that both exocytosis and
the calcium influx were inhibited by barium and
amiloride, with the same dose-response curve as for the
hyperpolarization-dependent calcium channel, whatever
the secretagogue, veratridine or AED. These experiments
suggest that the calcium influx is necessary for exocytosis
and that the mode of action of AED could be the transient
stimulation of hyperpolarization-dependent calcium channels through rapid surface charge screening, a phenomenon equivalent to a hyperpolarization at the level of
single channels (discussed by Cohen and Kerboeuf [96]).
In addition, the application of lysozyme, another secretagogue for Paramecium, activates a receptor-operated calcium conductance [63], a fact which also favors the
necessity of calcium entry for exocytosis.
3.6.2. … or release from internal stores?
Experiments showing that the chelation of external
calcium does not abolish membrane fusion lead to the
conclusion that a calcium influx was not strictly necessary
for exocytosis [111, 112], although it can facilitate the
exocytotic process [113]. Thus, the potential role of
calcium release from subplasmalemmal stores was examined. Caffeine, known as a potent agent to release calcium
from subplasmalemmal stores [114] by inhibiting refilling
through a sarco(endo)plasmic reticulum-like calciumATPase (SERCA) pump [104], appears to trigger trichocyst exocytosis [102, 111]. Raising intracellular free
calcium through intervention at the subplasmalemmal
stores, without triggering the normal sensors at the cell
surface, therefore appears sufficient to initiate the whole
exocytotic process. The strict parallel between current
recordings of cells triggered with AED and with caffeine
[111], as well as the visualization of localized calcium
transients by fluorescence calcium imaging, upon AED
stimulation in different external calcium concentrations
[97, 115], argue in favor of a primary role for calcium
release from internal stores in the induction of exocytosis.
The calcium influx would then be a secondary event to
refill the stores and/or to contribute to the cytosolic
calcium concentration necessary to trigger membrane
fusion. However, the actual kinetics of events induced by
‘regular’ triggering, i.e., without bypass by agents such as
caffeine and without chelation of external calcium (a
condition not encountered in Paramecium’s natural environment), still remains to be experimentally determined.
3.6.3. Will the answer come from the nd12-1 mutant?
All these conclusions are drawn from pharmacological
experiments using agents that may have side effects other
than the ones they are used for. In this context, genetic
arguments would be useful to corroborate some of the
hypotheses. The nd12-1 mutant is of considerable interest
in this context. Indeed, it is the only nd mutant having
roughly normal rosettes and connecting material, but
displaying an absence of the stimulus-dependent calcium
Vayssié et al.
influx correlated with the loss of exocytosis at nonpermissive temperature [105]. On the basis of this observation alone, it cannot be concluded that calcium influx
precedes exocytosis, but we can infer that the mutational
defect is upstream of both calcium influx and membrane
fusion. The possibility that, like the mutant tnd1 in
Paramecium caudatum which has a rosette [116], this
phenotype results from a defect in trichocyst matrix
expansion (thus exit from the cell) rather than in membrane fusion [117] can be excluded. Trichocysts isolated
from nd12-1 cells grown at non-permissive temperature
are able to perfectly decondense (J.Cohen, unpublished).
The mutation most likely affects the reception of the
signal or one of the steps of signal transduction that
precede both calcium influx and exocytosis. One way to
determine the sequence of calcium movements would be
the extensive study of nd12-1 cells using the pharmacological agents and the analytical methods already used on
the wild-type. For example, does AED application result
in calcium transients in the mutant? Does caffeine induce
such transients? If yes, does caffeine trigger exocytosis in
the mutant (i.e., bypass the mutational defect) and induce
a calcium influx?
Cloning and sequencing the ND12 gene could provide
critical insights into the order and respective roles of
calcium movements during exocytosis.
4. Conclusions and perspectives
Trichocyst exocytosis in Paramecium is the regulated
secretory pathway that has been the most extensively
approached by genetics. Mutants have been obtained that
affect all steps, from the early ones which should be in
common with the constitutive pathway, and in this respect
probably equivalent to some SEC mutants of yeast, up to
the very last ones which correspond to terminal exocytotic
membrane fusion in response to stimulation. Two main
properties of the system make its study very attractive: 1)
the crystalline and dynamic nature of the contents provides a good model for ordered molecular assembly of
multiple polypeptides; and 2) like the cortical granules in
oocytes and like the active subpopulation of synaptic
vesicles in nerve terminals, the trichocysts are normally
docked at the plasma membrane in a frozen step just prior
to membrane fusion. The only step triggered by the
stimulation, through signal reception and transduction, is
membrane fusion. This final exocytotic event is thus
amenable to experimental study in Paramecium without
interference from anterior steps.
For a decade now, molecular biology of this organism
has been actively developed so that we have insights into
the nature of the secreted products and, most recently, into
some of the genes involved in membrane fusion. It should
only be a matter of time and effort to have a complete
overview of the genes controlling trichocyst secretion.
Molecular genetics of regulated secretion in Paramecium
285
Will this approach allow us to identify key molecules not
yet discovered in mammalian exocytotic systems, or
instead reveal specialized molecules developed by ciliates
and other alveolates? Whether or not Paramecium turns
out to really be a ‘swimming neuron’, we approach the
next millennium with hopes of studying new molecules
and functions that could open fruitful avenues to combat
cell invasion by Paramecium’s deadly cousins, the apicomplexan parasites.
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Note added in proof
The following reference has been accepted: Galvani A.,
Sperling L., Regulation of secretory protein gene expression in Paramecium: role of the cortical exocytic sites,
Eur. J. Biochem. (2000), in press.
Acknowledgments
We thank Janine Beisson for critical reading of the manuscript
and acknowledge grant no. 96024 from the Centre National de la
Recherche Scientifique ‘Biologie cellulaire: du normal au
Pathologique’ and a grant from the Ministère de l’Education
National, la Recherche et la Technologie ‘Programme de Recherche Fondamentale en Microbiologie, Maladies Infectieuses et
Parisitaires’.
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