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

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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

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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

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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-

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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.

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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-

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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

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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.

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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.

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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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