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MOLECULAR AND CELLULAR BIOLOGY, July 2009, p. 3605–3622 Vol. 29, No. 130270-7306/09/$08.00�0 doi:10.1128/MCB.01592-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Novel Types of Ca2� Release Channels Participate in the SecretoryCycle of Paramecium Cells�†

Eva-Maria Ladenburger,* Ivonne M. Sehring, Iris Korn, and Helmut PlattnerDepartment of Biology, University of Constance, 78457 Constance, Germany

Received 10 October 2008/Returned for modification 20 November 2008/Accepted 14 April 2009

A database search of the Paramecium genome reveals 34 genes related to Ca2�-release channels of theinositol-1,4,5-trisphosphate (IP3) or ryanodine receptor type (IP3R, RyR). Phylogenetic analyses show thatthese Ca2� release channels (CRCs) can be subdivided into six groups (Paramecium tetraurelia CRC-I toCRC-VI), each one with features in part reminiscent of IP3Rs and RyRs. We characterize here the P.tetraurelia CRC-IV-1 gene family, whose relationship to IP3Rs and RyRs is restricted to their C-terminalchannel domain. CRC-IV-1 channels localize to cortical Ca2� stores (alveolar sacs) and also to theendoplasmic reticulum. This is in contrast to a recently described true IP3 channel, a group II member (P.tetraurelia IP3RN-1), found associated with the contractile vacuole system. Silencing of either one of theseCRCs results in reduced exocytosis of dense core vesicles (trichocysts), although for different reasons.Knockdown of P. tetraurelia IP3RN affects trichocyst biogenesis, while CRC-IV-1 channels are involved insignal transduction since silenced cells show an impaired release of Ca2� from cortical stores in responseto exocytotic stimuli. Our discovery of a range of CRCs in Paramecium indicates that protozoans alreadyhave evolved multiple ways for the use of Ca2� as signaling molecule.

Ca2� is an important component of cell activity in all organ-isms, from protozoa to mammals. Thereby Ca2� may originatefrom the outside medium and/or from internal stores (7, 18).Ca2� release from internal stores is mediated by various Ca2�

release channels (CRCs), of which the inositol-1,4,5-trisphos-phate receptor (IP3R) and ryanodine receptor (RyR) familieshave been studied most extensively (8, 9, 29, 63). IP3Rs andRyRs have been identified in various metazoan organisms (re-viewed in references 9, 28, and 104). According to these re-views, there exist three genetically distinct isoforms of eachreceptor type in mammals and orthologues have been identi-fied in various nonmammalian vertebrates, e.g., frogs, chick-ens, and fish. RyRs and IP3Rs were also cloned and sequencedin the invertebrates Drosophila melanogaster and Caenorhabdi-tis elegans, which possess one copy of each receptor type.

Functional evidence for Ca2� release in response to ryano-dine or IP3 receptor agonists has been described in severalunicellular systems. Treatment of permeabilized Plasmodiumchabaudi parasites with IP3 results in Ca2� release, which isinhibited by the IP3 receptor antagonist heparin (69). Anotherapicomplexan parasite, Toxoplasma gondii, responds to ago-nists and antagonists of both, ryanodine and IP3 receptors, bymediating increases in intracellular Ca2� concentration([Ca2�]i) (56). Stimulation of Trypanosoma cruzi with carba-chol results in increased [Ca2�]i and IP3 (59). IP3 and cyclicADP-ribose induces Ca2� release in Euglena gracilis micro-some fractions in a dose-dependent manner (61). In the giantalgae Chara corallina and Nitrella translucens, IP3 produces

action potentials involving increased [Ca2�]i (93). Treatmentof vacuolar membrane vesicles from Candida albicans with IP3

results in Ca2� release, blocked by heparin and ruthenium red(14). IP3 generates and maintains a Ca2� gradient in the hy-phal tip of Neurospora crassa and the IP3-sensitive channelshave been reconstituted and characterized with the planarbilayer method (87). In summary, these publications suggestthat IP3-dependent signaling pathways are conserved amongunicellular organisms, including protozoa.

Despite these data, the molecular characterization of IP3 orryanodine receptors in low eukaryotes is currently a challengesince the identification of orthologues has not been possiblethus far, probably because of evolutionary sequence diver-gence (66). Traynor et al. (96) identified an IP3 receptor-likeprotein, IplA, in Dictyostelium discoideum, which possesses re-gions related to IP3R sequences, but thus far no evidence forIP3 interaction exists. We have recently described an IP3R inthe ciliated protozoa Paramecium tetraurelia (referred to hereas P. tetraurelia IP3RN) (53), with features characteristic ofmammalian IP3Rs in terms of topology and ability for IP3

binding. The expression level of P. tetraurelia IP3RN is modu-lated by extracellular Ca2� concentrations ([Ca2�]o) and im-munofluorescence studies reveal an unexpected localization tothe contractile vacuole complex (CVC), the major organelleinvolved in osmoregulation (2). The ionic composition of thecontractile vacuole fluid by ion-selective microelectrodes (91)suggests that the organelle plays a major role in expelling anexcess of cytosolic Ca2�. Therefore, these IP3Rs may heremediate a latent, graded reflux of Ca2� for fine-tuning of[Ca2�]i and thus serve [Ca2�] homeostasis (53).

Besides [Ca2�] homeostasis, the Paramecium cell has toregulate a variety of well-characterized processes (75). Thisincludes exocytosis of dense-core secretory vesicles (tricho-cysts) (71, 74, 99). Each cell possesses up to 1,000 trichocystsattached to the cell membrane. Their contents can be extruded

* Corresponding author. Mailing address: Department of Biol-ogy, University of Constance, P.O. Box 5560, 78457 Constance,Germany. Phone: 49-7531-884230. Fax: 49-7531-882245. E-mail:[email protected].

† Supplemental material for this article may be found at http://mcb.asm.org/.

� Published ahead of print on 20 April 2009.

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synchronously in response to natural stimuli, i.e., predators(34, as confirmed by Knoll et al. [49]), to artificial polyaminesecretagogues such as aminoethyldextran (AED) (78), to caf-feine (48) or to the ryanodine substitute, 4-chloro-meta-cresol(4-CmC) (46). Their expulsion strictly depends on Ca2� (10)and is accompanied by an increase of intracellular [Ca2�]i (24,47). This Ca2� signal originates from rapid mobilization ofcortical stores, the alveolar sacs (33, 64, 74), superimposed byCa2� influx (46, 72). It thus represents a SOC-type mechanism(SOC, store-operated Ca2� entry) known from mammaliansystems (81).

Upon exocytosis stimulation �60% of their total Ca2� isreleased from alveolar sacs (33). These are Ca2� stores (90)represented by flat membrane compartments tightly attachedat the cell membrane surrounding each trichocyst docking site.They possess a SERCA-type pump located at the membranefacing the cell center (36, 37) and a luminal high-capacity/low-affinity CaBP of the calsequestrin type (73). Thus far, Ca2�

release channels of these stores were identified only indirectlyas cells respond by exocytosis to the RyR activators caffeine(54, 48) and 4-CmC (46). However, an involvement of con-served RyRs has remained questionable as ryanodine is notable to activate Ca2� release from alveolar sacs, as is the casewith IP3 (54). Therefore, one of the most intriguing questionsis the elucidation of the molecular nature of the channelsmediating Ca2� release from alveolar sacs upon stimulatedexocytosis.

In the present work we describe a novel family of CRCs (P.tetraurelia CRC-IV-1), whose members display several proper-ties of the channels postulated above. In detail, the identifiedCRC-IV-1 channels localize to the alveolar sacs. Functionaland fluorochrome analyses after gene silencing reveal that theyare essential for mediating Ca2� release and exocytosis inresponse to AED, caffeine, or 4-CmC. Their classification as“novel” CRC type is based on a restricted relationship to theC-terminal channel domains of IP3Rs and RyRs. The overallsize and the number of putative transmembrane domains re-semble IP3Rs, but N-terminal parts of CRC-IV-1 channels donot show any conservation, such as an IP3-binding domain.Therefore, CRC-IV-1 channels represent distant relatives ofIP3Rs and RyRs and may belong to an ancestral Ca2� signalingpathway.

MATERIALS AND METHODS

Paramecium cultures. P. tetraurelia wild-type stocks 7S and d4-2 derived fromstock 51S (89) were cultivated at 25°C in a decoction of dried lettuce (SMmedium) supplemented with 0.4 �g of �-sitosterol/ml. Cells were grown mon-oxenically by inoculation with Enterobacter aerogenes or in a sterile syntheticmedium (41).

Computational analysis. BLAST searches were performed according to themethod of Altschul et al. (3) by using either the NCBI database (http://blast.ncbi.nlm.nih.gov), the Paramecium genome database (http://paramecium.cgm.cnrs-gif.fr) or the transporter protein analysis database TransportDB (http://www.membranetransport.org). For databases used in addition for other genomes seeTable S4 in the supplemental material. Protein alignments were performed withCLUSTAL W (94). Phylogenetic and molecular evolutionary analyses were carriedout by using MEGA version 3.0 (50). Predictions of membrane topologies wereperformed with HMMTOP 2.0 (97) or with the TopPred II algorithm (19).

Determination of CRC-IV-1 sequences. To determine macronuclear sequencesof CRC-IV-1a, we screened a genomic library of P. tetraurelia according to themethod of Keller and Cohen (42). Full-length sequences of CRC-IV-1a andCRC-IV-1b were amplified with the AccuTaq LA DNA polymerase (Sigma,Munich, Germany) using 50 ng of macronuclear DNA as a template (for primers,

see Table S1 in the supplemental material). PCRs were carried out in 30 cyclesof 95°C for 30 s, 54°C for 30 s, and 68°C for 12 min. The complete open readingframe (ORF) of CRC-IV-1a and parts of the ORF of CRC-IV-1b were identifiedby amplifying mRNA sequences by reverse transcription-PCR (RT-PCR). Prep-aration of RNA and cDNA synthesis was performed as described previously (53).PCR amplification of cDNAs occurred with the Advantage 2 PCR enzymesystem (Clontech, Palo Alto, CA) (for primers, see Table S1 in the supplementalmaterial), and reactions were carried out in 35 cycles of 95°C for 30 s, 53°C for20 s, and 68°C for 120 s. PCR-amplified macronuclear DNA and cDNA frag-ments were cloned in the pCRII-TOPO cloning system (Invitrogen, Carlsbad,CA) and analyzed by sequencing (Eurofins MWG Operon, Martinsried, Ger-many).

Abs. To produce antibodies (Abs) to CRC-IV-1, we amplified macronuclearDNA corresponding to residues P2458 to H2556 of CRC-IV-1a by PCR (forprimers, see Table S1 in the supplemental material) and cloned the fragment intothe pET16b expression vector (Novagen, Madison, WI). Purification of theHis-tagged P2458-H2556 peptide, as well as Ab production and purification, wasperformed as described previously (53).

The affinity purified Abs (R772 and R883) were used at concentrations of 12�g/ml (R772) or 5 �g/ml (R883) in immunofluorescence studies and 0.25 �g/mlin Western blots. The polyclonal mouse Ab directed against protein disulfideisomerase (PDI) from P. tetraurelia was raised against a polypeptide correspond-ing to N-terminal amino acids L33-E145 of PDI1-1 (accession no. CAD99202).The serum obtained after repeated injections of the antigen was directly used;either diluted 1:1,000 for Western blots or 1:100 for immunostainings. Absagainst trichocyst matrix protein 1 (TMP1) and trichocyst matrix protein 4(TMP4) were generously provided by Linda Sperling (Centre de GenetiqueMoleculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette,France) and used diluted 1:600 (TMP1) or 1:800 (TMP4) for immunostainingsand 1:4,000 (TMP1) or 1:8,000 (TMP4) for Western blots, respectively. Thepolyclonal rabbit Ab directed against V-type H�-ATPase is described in Wass-mer et al. (101) (there designated as “anti-a1-1” [P178-S328]) and was used at aconcentration of 12 �g/ml in immunostainings. The Ab against �-tubulin was amonoclonal mouse Ab (clone DM1A; Sigma-Aldrich, Schnelldorf, Germany),which was used diluted 1:200 in immunostainings and 1:4,000 in Western blots.

Cell harvesting, protein preparation, and Western blots. Cells were harvestedby centrifugation (2 min; 100 � g), washed twice with PIPES buffer (pH 7.0)containing 5 mM PIPES (piperazine-1,4-bis[2-ethanesulfonic acid]), 1 mM KCland 1 mM CaCl2. Aliquots (100 �l) were frozen in liquid nitrogen. Samples werethawed in the presence of a protease inhibitor cocktail (complete; Roche Diag-nostics, Basel, Switzerland), 400 �l of methanol and 100 �l of chloroform. Afterthe addition of 300 �l of double-distilled H2O probes were centrifuged for 5 minat 4,500 � g, and the aqueous phase was removed, followed by adding 300 �l ofmethanol. After vortexing and a second centrifugation step (5 min, 4,500 � g),the supernatant was removed, and the pellet was air dried. Pellets were dissolvedin 2.5% sodium dodecyl sulfate (SDS) and complemented with tenfold-concen-trated sample buffer (0.1 M Tris-HCl [pH 8], 50% �-mercaptoethanol, 32%glycerol). Proteins (10 �g/lane) were separated by SDS-polyacrylamide gel elec-trophoresis (PAGE) according to the method of Laemmli on 12% polyacrylam-ide gels and transferred onto nitrocellulose membranes. Membranes wereblocked with 5% nonfat dry milk dissolved in Tris-buffered saline plus Tween 20(10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20 [pH 8]) and incubated for 1 hwith primary Abs. Detection occurred by goat anti-rabbit or goat anti-mouseimmunoglobulin G (IgG) coupled to horseradish peroxidase (1:25,000; Dianova,Hamburg, Germany); luminescence was visualized with an ECL Western blottingsystem (Amersham, Freiburg, Germany).

For CRC-IV-1 detection in Western blots, cells were injected in boiling 10%SDS containing protease inhibitors. These contained 15 �M pepstatin A and 42�M Pefabloc (Serva, Heidelberg, Germany), 100 �M leupeptin, and 28 �M E64(Biomol, Hamburg, Germany), 75 mU of aprotinin/ml, 10 �M chymostatin, and10 �M antipain (Sigma, Munich, Germany). After completing with tenfold-concentrated sample buffer, samples were directly subjected to SDS-PAGE.

Immunolocalization and affinity labeling studies. Immunolabeling with Absagainst CRC-IV-1 (R772 and R883), PDI, and V-type H�-ATPase was per-formed as described previously (53). Labeling of trichocyst matrix proteins and�-tubulin was performed by permeabilization of knockdown cells for 2 min with1% Triton X-100 in PHEM buffer [60 mM PIPES, 25 mM N-(2-hydroxyethyl)-piperazine-N�-2-ethanesulfonic acid (HEPES), 10 mM EGTA (ethylene glycoltetraacetic acid), 2 mM MgCl2 (pH 6.9)], followed by a fixation period of 10 minin 2% formaldehyde dissolved in PHEM buffer. Formaldehyde was removed bytwo washing steps with modified Tris-buffered saline (mTBS) containing 10 mMTris-HCl, 15 mM NaCl, 0.1% Tween 20, and 3% bovine serum albumin (pH 7.4).Then cells were incubated for 30 min with primary Abs diluted in mTBS and

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after two washing steps with secondary Abs diluted in mTBS. We applied AlexaFluor 488 or 594 F(ab�)2 fragments of goat anti-rabbit IgG, or Alexa Fluor 594F(ab�)2 fragments of goat anti-mouse IgG (H�L; Molecular Probes, Eugene,OR) diluted 1:150 in phosphate-buffered saline–1% bovine serum albumin. Fi-nally, cells were washed twice with mTBS and mounted with Mowiol for confocalmicroscopy. Membrane staining with 3,3�-dihexaoxacarbocyanine iodide(DiOC6; Sigma) was performed after fixation at a final concentration of 0.1 �gof DiOC6 per ml of phosphate-buffered saline for 1 h.

Conventional fluorescence microscopy was carried out on an Axiovert 100TV(Carl Zeiss, Jena, Germany) equipped with fluorescein isothiocyanate (FITC)-Filterset 9 and with a ProgRes C10 plus camera (Jenoptik, Jena, Germany).Images were captured by using ProCa 2.0 software (Carl Zeiss). Confocal mi-croscopy was carried out with a LSM 510 META using a �63 Plan-Apochromat(NA � 1.4) objective (both Carl Zeiss). Images were processed by using LSMimage browser (Carl Zeiss) and further edited with Adobe Photoshop (AdobeSystems, San Jose, CA).

Immuno-electron microscopy localization. The method applied is as specifiedby Kissmehl et al. (45). In brief, cells were injected into 8% formaldehyde plus0.1% glutaraldehyde (pH 7.2) at 0°C, with a quenched-flow device, and processedby the “progressive lowering of the temperature” method. We reduced thetemperature stepwise, with increasing ethanol concentrations. This was followedby LR Gold embedding and UV polymerization at 35°C. CRC-IV-1 specificAbs (R883) have been used for localization by protein A-gold conjugated to5-nm gold (pA-Au5) in a Zeiss electron microscope, EM10.

Gene silencing constructs. Tandem constructs representing both isoforms,either of the IP3RN or of the CRC-IV-1 gene families, were cloned into thedouble T7-promoter plasmid pL4440 described by Timmons and Fire (95), heredesignated as pPD vector. An 1,170-bp cDNA fragment of IP3RN-1 was ampli-fied by PCR and cloned with XhoI and PstI. IP3RN-2 specific sequences wereadded by introducing a 1,019-bp PCR fragment using MluI and PstI. For CRC-IV-1 receptors, a 872-bp PCR-amplified cDNA fragment of CRC-IV-1a and a945-bp cDNA fragment of CRC-IV-1b (for primers, see Table S1 in the supple-mental material) were first subcloned in the pCR2.1 TOPO vector (Invitrogen).Cloning into the pPD vector was performed with XhoI and SpeI for CRC-IV-1asequences, followed by insertion of CRC-IV-1b sequences using XhoI and KpnI.The plasmids were designated pPD-N1N2 (IP3RN specific) or pPD-C1C2 (CRC-IV-1 specific). As a negative control (pPD-GFP), the coding sequence of greenfluorescent protein (GFP) derived from the pPXV-GFP vector (35) was clonedby using KpnI.

Gene silencing by feeding. pPD constructs containing open reading frames ofeither GFP (pPD-GFP, negative control), nd7 (non-discharge 7 gene [88], pos-itive control) or parts of both isoforms of IP3RN (pPD-N1N2) and of CRC-IV-1(pPD-C1C2), respectively, were transformed into E.coli strain HT115 (DE3).Single colonies were picked and bacteria were grown to an optical density at 600nm of 0.25 in lysogeny broth (LB) medium supplemented with tetracycline (12�g/ml) and ampicillin (100 �g/ml) and double-stranded RNA synthesis wasinduced in the presence of 0.5 mM IPTG (isopropyl-�-D-thiogalactopyrano-side). After 3 h of induction, bacteria were harvested by centrifugation (15min at 4,000 � g), and the LB medium was completely removed. Feedingsolutions were prepared by resuspending bacteria in SM medium supple-mented with ampicillin (100 �g/ml), �-sitosterol (0.4 �g/ml), and 0.5 mMIPTG. After adjusting the optical density at 600 nm to 0.2, feeding solutionswere applied to Paramecium cell cultures after starving for 3 h in PIPESbuffer (pH 7.0; 5 mM PIPES, 1 mM KCl, 1 mM CaCl2).

Cells were daily counted and transferred to freshly prepared feeding solutionsby adjusting the cell number to 50 cells per ml. For single cell analyses, Para-mecium cells were separated in depression wells containing 100 �l of feedingsolution.

Analyses of RNA from silenced cells. Total RNA from silenced cells wasprepared by either by using the High-Pure RNA isolation kit (Roche Diagnos-tics) or, for single cell analyses, the RNeasy micro kit (Qiagen, Hilden, Germany)according to the manufacturers’ protocols. cDNAs were obtained by using theQuantiTect reverse transcription kit (Qiagen).

Amplification and quantification of cDNAs were performed by real-time PCRin the Bio-Rad iCycler iQ real-time PCR detection system. Reactions werecarried out in a final volume of 25 �l using 2 �l of cDNA, 2� iQ SYBR GreenSupermix (Bio-Rad,; Munich, Germany), and 12.5 pmol each of forward andreverse primers (see Table S1 in the supplemental material). Thermal cycling wasinitiated with a first denaturation step of 3 min at 95°C, followed by 45 cycles of95°C for 20 s, 54°C for 20 s, and 70°C for 30 s; the amplification fluorescence wasread at 70°C at the end of the cycle. All primer pairs were tested with macro-nuclear DNA and cDNA, and in all cases a single amplicon of the appropriatemelting temperature and size was verified by dissociation curves and gel elec-

trophoresis. PCR amplification data were collected with the Bio-Rad iCycler iQreal-time detection system software and analyzed using the threshold cycle (CT)relative quantification method as described by Livak and Schmittgen (55). Fornormalizing the data we used actin 1-1 (act1-1; accession no. AJ537442) as thereference gene, and data were calculated using the relative expression withrespect to act1-1 as 100%.

Physiological tests. To assay the sensitivity of silenced cells to different[Ca2�]o, cells were incubated in feeding solutions supplemented with 1 mMEGTA to yield different [Ca2�]o values adjusted by adding 1.1, 1, or 0.85 mMCaCl2. Free [Ca2�]o was calculated according to the method of Patton et al. (70)using the MaxChelator program Winmaxc v.2.40. Cells were counted daily, anddivision rates were determined by calculating the number of cell fissions per day.Capability of trichocyst exocytosis was mimicked by adding an excess of saturatedpicric acid (79). The pumping cycles of contractile vacuoles were determined bymeasuring the time between contractions of the central vacuoles of cells con-tained in a microdrop overlaid with paraffin oil.

Ca2� fluorochrome analyses. Cells were immobilized and loaded by microin-jection with the Ca2� fluorochrome, Fura Red, as previously described (47).Intracellular concentration of Fura Red was �50 �M. For excitation, two wave-lengths were used (excit � 440/490 nm), and the ratio of emission (emiss � 650nm) at both wavelengths was calculated. This allowed the analysis of Ca2�

independently of fluorochrome concentration in the cell and of cell shapechanges. The area evaluated, 3 by 10 �m, was oriented parallel to the cell surfaceat the cortical region. The ratio analysis was then transformed into f/f0 values,which means that any fluorescence readings during stimulation (f) were referredto the reading before stimulation (f0). The f/f0 ratios were expressed as apparent[Ca2�]i increase values, starting from 1 as a representative resting value of[Ca2�]i. Measurements were made at a Zeiss ICM 405 microscope.

All trigger agents were diluted in 5 mM PIPES (pH 7.0). The actual concen-tration of the trigger at the cell surface was estimated from the dilution aspreviously described (47). The final extracellular concentrations of AED (78),caffeine (48), and 4-CmC (46) were 2 �M, 50 mM, and 0.5 mM, respectively. Lowvalues of [Ca2�]o were achieved by adding the ultrafast Ca2� chelator BAPTA(final concentration, 1 mM) to the trigger solution to yield a [Ca2�]o of �30 nM,i.e., slightly below [Ca2�]i at rest (�65 nM [47]). This allowed us to monitorselectively the Ca2� signal resulting from the release from cortical stores andundisturbed by Ca2� influx.

Accession numbers. The ParameciumDB accession number for CRC-IV-1a isPTETG13800001001. CRC-IV-1b sequences were submitted to the EMBL da-tabase and are available under accession number BN001236.

RESULTS

Identification of genes related to mammalian IP3 and ryan-odine receptors. We screened the P. tetraurelia genome (4, 5)for identifying putative genes related to mammalian intracel-lular Ca2� release channels of the IP3 or ryanodine receptortype. In detail, we performed a homology-based databasesearch with sequences corresponding to channel domains ofknown IP3Rs and RyRs, the RIH (RyR and IP3R homology)consensus sequence and the IP3-binding domain of Mus mus-culus IP3R1. After authentication of the received sequences byBLASTP analyses using the NCBI database and InterProScandomain analyses (107), the Paramecium genome yields 34genes coding for possible CRCs (Fig. 1). Phylogenetic analysesreveal that the 34 candidates can be divided in six groups (Fig.1A), each showing characteristic features regarding their rela-tionship to IP3Rs or RyRs. As determined by BLAST analyses(3), the positions of conserved regions of individual CRCs (Fig.1B; for details, see Table S3 in the supplemental material)mostly overlap within one group, thereby confirming the dis-position of the 34 genes into six groups.

At a first glance, regarding the size of the CRCs identified inP. tetraurelia, all of them seem to be more related to IP3Rs thanto RyRs, since none of them reach molecular weights knownfrom metazoan RyRs consisting of roughly 5,000 amino acids(29, 104). However, similarities to RyRs are restricted to C-

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terminal parts of the proteins, and only N-terminal parts ofgroup I members share considerable similarity to RyRs (Fig.1B). The relationship to IP3Rs is most pronounced in group Iand II proteins, where a central RIH domain could be identi-fied in all group members. CRCs of group III share homolo-gous regions to IP3Rs covering almost the complete receptorsequences, but none of them possess a RIH domain, whichagain emerges in some members of group V CRCs. In contrastto group II proteins, the RIH domain of group V membersappears in N-terminal parts of the proteins. Group IV proteinsshow an aberrant architecture, as IP3R- and RyR-related re-gions seem to be restricted to a highly conserved channeldomain so that the N-terminal parts of these proteins do not fitthe known pattern of IP3Rs or RyRs (Fig. 1B).

Molecular characterization of CRC-IV-1 channels. Previ-ously two partial sequences of CRC genes could be identifiedin a pilot genome survey of P. tetraurelia (21), implying twoclones, M22Ho1r and M24E11u(rc), containing sequences ho-

mologous to IP3Rs. One gene (P. tetraurelia IP3RN-1), repre-sented by M24E11u(rc), was recently characterized in detail(53) (accession no. CR932323), while the present work envis-ages unraveling the gene represented by M22Ho1r. First, wehave screened an indexed genomic library (42) in the lab ofJean Cohen (CNRS, France), allowing us to identify 11 clonescovering 8,892 bp of the gene. Residual sequence informationwas also obtained from the still ongoing Paramecium genomeproject organized by Genoscope (http://www.genoscope.cns.fr). The cDNA sequences were cloned and analyzed, resultingin an ORF with 8,991-bp coding for a protein with 2,997 aminoacids and a calculated molecular mass of 351 kDa. Comparisonof the genomic sequence with their cDNA equivalent resultedin two introns each of 25 bp (Fig. 2A). Based on the relation-ship to the other Paramecium CRCs (Fig. 1), we could classifythis gene as a member of group IV CRCs belonging to a familyof two closely related isoforms. The gene was designated CRC-IV-1a and the related isoform as CRC-IV-1b. Both isoforms

FIG. 1. Identification of 34 Paramecium CRCs related to IP3Rs and RyRs. (A) Evolutionary relationship of putative CRC proteins fromParamecium. Predictions from multiple sequence alignments are shown in a neighbor-joining tree with 1,000 bootstrap replicates generated withthe MEGA version 3.0 program (50). Sequences representing three different metazoan IP3 receptors are from Mus musculus (identified asMmIP3R1 in the figure; accession no. NP_034715.2), Drosophila melanogaster (DmIP3R, accession no. BAA14399.1), or Caenorhabditis elegans(CeITR1, accession no. NP_001023173). Accession numbers of the Paramecium sequences are summarized in Table S2 in the supplementalmaterial. Bootstrap support values are given at the branches, and evolutionary distances are indicated by the scale bar below. “Sc” denotes scaffoldnumbers according to macronuclear DNA sections as designated in the P. tetraurelia genome database (http://paramecium.cgm.cnrs-gif.fr).(B) Sequence analysis of putative CRCs from P. tetraurelia and their relationship to RyRs (represented by CeRyR, accession no. BAA08309.1) andIP3Rs (represented by MmIP3R1, accession no. above). Gray bars represent regions homologous to IP3Rs; regions homologous to RyRs are shadedin green. Positions of flanking residues of putative channel domains are highlighted in red and IP3-binding domains are highlighted in blue.Conserved regions were determined by BLAST analyses (3) (dark gray bars) and extended by using the BLAST 2 SEQUENCES tool (92) (lightgray bars and green bars). Positions of flanking residues of conserved CRC sequences, as well as corresponding e-values are outlined in Table S3in the supplemental material. RIH, RyR and IP3R homology domain according to Ponting (80); Sc, scaffold number.


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share 83.3% identity on DNA and 89.7% identity on aminoacid level. Sequence comparisons with the Paramecium data-base are shown in Fig. S1 in the supplemental material.

According to their molecular topology, group IV members,CRC-IV-1a and CRC-IV-1b, are conserved regarding theirC-terminal domains, which show high agreement with corre-sponding regions of mammalian IP3Rs (Fig. 2B). Analysis byKyte-Doolittle scales (52) reveal that CRC-IV-1a and CRC-IV-1b (data not shown) possess six hydrophobic stretcheswithin this region (Fig. 2C). Thus, they resemble IP3R channeldomains, which consist of six transmembrane spanning seg-ments (TMD1 to TMD6) with the pore region between TMD5

and TMD6 (62). Two parts of the CRC-IV-1 C termini couldalso be aligned with sections of RyRs. One part in each isoform(CRC-IV-1a I2240-V2562 and CRC-IV-1b I2247-F2585) over-laps with a region responsible for RyR activation by 4-CmC(26, 27) (sequence alignments are shown in Fig. S2 in thesupplemental material), the other part (CRC-IV-1a D2744-P2965 and CRC-IV-1b D2748-P2968) matches with RyR pore-forming segments. Focusing on the pore region, sequencealignments show that the Paramecium CRC-IV-1 channels, aswell as the recently described P. tetraurelia IP3RN-1 receptor(53), and its closely related isoform P. tetraurelia IP3RN-2 (seeFig. 1) are related rather to RyRs than to IP3Rs. As shown in

FIG. 2. Molecular structure of the Paramecium CRC-IV-1 channels. (A) Schematic representation of both isoforms of CRC-IV-1 genes fromP. tetraurelia. Start (�1) and stop (�9044) codons of CRC-IV-1a were determined by RT-PCR, as were the positions of introns (triangles), whileCRC-IV-1b was determined on the basis of homology. (B) Schematic representation of CRC-IV-1a. The putative channel domain (L2606 to D2880)is shaded in gray; “Ab” denotes the antigenic region (P2457 to H2556) used to raise polyclonal Abs. Alignments showing CRC-IV-1a regionshomologous to RnIP3R3 and M. musculus RyR1 are listed below, as well as to P. tetraurelia CRC-IV-1b and three orthologues identified in T.thermophila (TTHERM_00138560, TTHERM_00762850, and TTHERM_00762860). Positions of residues referring to CRC-IV-1a sequences arehighlighted in boldface type above the traces (P. tetraurelia [Pt]), whereas positions of residues referring the homologous counterparts (Rattusnorvegicus [Rn], M. musculus [Mm], and T. thermophila [Tt]) are indicated with the corresponding e-values below. (C) Hydropathy profile of thechannel domain of CRC-IV-1a (F2580 to H2920) by Kyte-Doolittle scales (52) reveals six highly hydrophobic segments. Putative positions oftransmembrane and pore regions (I to VI, P) were predicted by using the TopPred II algorithm (19). (D) Multiple sequence alignment of putativepore regions (highlighted in red) with adjacent transmembrane segments (highlighted in blue) of Paramecium CRCs IP3RN-1 (accession no.CAI39149), IP3RN-2 (accession no. CAI39148), CRC-IV-1a, and CRC-IV-1b in comparison to metazoan IP3Rs (Mus musculus IP3R1, Drosophilamelanogaster IP3R, and Caenorhabditis elegans ITR) and RyRs (Mus musculus RyR1, D. melanogaster RyR [accession no. BAA04212], and C.elegans RyR [accession no. BAA08309]). Residues that are identical are shaded in black; similar residues are shaded in gray. The amino acidshighlighted in yellow within the pore segment correspond to putative selectivity filter regions (11). In RyR sequences the glutamine critical forryanodine interaction is marked in green (100). Predictions of membrane topologies were performed with HMMTOP 2.0 according to the methodof Tusnady and Simon (97).


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Fig. 2D, the Paramecium CRCs, just like metazoan RyRs, lackthe extended luminal loop region between TMD5 and thepore, as it occurs in metazoan IP3Rs. The putative selectivityfilter motif GGGI/VGD within the pore regions of RyRs andIP3Rs (9, 11) is conserved in CRC-IV-1 channels, whereas theglutamine present in C-terminal transmembrane segments ofRyRs critical for ryanodine interaction (100) is not present inCRC-IV-1 channels.

N-terminal parts of CRC-IV-1 proteins are less conservedand only sequence searches of the genome of the relatedciliate Tetrahymena thermophila (23) revealed three genes(TTHERM_00138560 [GenBank accession no. XP_001019838.2], TTHERM_00762850 [XP_001025343.1], TTHERM_00762860 [XP_001025344.1]), which share homologous re-gions covering the complete CRC-IV-1a protein (Fig. 2B).This suggests that these kinds of CRCs might be ciliatespecific. Thus far, we were not able to identify orthologuesin any other ciliate using the Ciliate Ortholog Database(http://oxytricha.princeton.edu/COD/index.html).

CRC-IV-1 channels are localized in the endoplasmic retic-ulum (ER) and in the alveolar sacs. To investigate the subcel-lular localization of CRC-IV-1 channels, we raised polyclonalAbs (R772 and R883) against a recombinant peptide compris-ing residues P2457-H2556 of CRC-IV-1a. This region shares85% identity (e � 5 � 1025) to the corresponding region inCRC-IV-1b, suggesting that both members of CRC-IV-1 chan-nels are recognized by the Abs. Considering any possible cross-reactions of the Abs, sequence analyses were performed. Thisreveals that the CRC-IV-1a peptide P2457-H2556 shares nosignificant similarity with any other CRC proteins. Only someadditional group IV members show weak conservation. In de-tail, identities are as follows: CRC-IV-4b(Sc3A), identities19/63 (30%) corresponding to e � 4.5; CRC-IV-4a(Sc6), iden-tities 15/37 (40%), e � 5.9; CRC-IV-2(Sc9B), identities 16/37(43%), e � 0.18; CRC-IV-3b(Sc24), identities 15/44 (34%),e � 2.0; and CRC-IV-3a(Sc62), identities 16/44 (36%), e � 0.7.Therefore, the Abs used are expected to be highly selective forCRC-IV-1a and -1b.

In Western blot analyses, affinity-purified R883 Abs recog-nize the CRC-IV-1a peptide P2457-H2556 with high affinity(Fig. 3A) and detect a high-molecular-weight band in Parame-cium whole-cell homogenates, which seems to be stabilizedthrough the presence of divalent cations (Fig. 3B). Immuno-fluorescence labeling with the same Ab results in a strongstaining of a network throughout the cell (Fig. 3D) and ofregularly arranged plate-shaped structures in the cell cortex(Fig. 3C) clearly representing alveolar sacs for reasons speci-fied below and in the Discussion. Colocalization studies with

FIG. 3. CRC-IV-1 is localized in the ER and in alveolar sacs.(A) Purified Abs against CRC-IV-1 (R883) recognize the polypeptidecorresponding to CRC-IV-1a residues P2457 to H2556 with high af-finity (lanes 2, 3, and 4) in immunoblots, whereas the preimmuneserum (PIS) yields no signal (lane 5). Lane 1 (COOM), Coomassieblue staining of purified polypeptide (2.5 �g) used for immunization.

(B) Western blot analysis of Paramecium whole-cell homogenates us-ing Ab R883 reveals two bands of �250 and �30 kDa (arrows). Thediscrepancy from the value expected may be due to partial degrada-tion. Samples were prepared in the presence (�) or absence () ofEDTA. (C and D) Confocal images of a cell stained with anti-CRC-IV-1 Abs (R883; red channel) and with DiOC6 (green channel) showcolocalization of both markers in the ER, but not in the alveolar sacs,which were labeled only with R883 Abs. (C and D) Confocal sliceswere taken from surface (C) and median sections (D) of the cell. oc,oral cavity. Scale bars, 10 �m.


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DiOC6, a marker for the ER applicable to Paramecium (82),show that the DiOC6-labeled meshwork overlaps largely withthe R883 staining in the cell interior (Fig. 3D), suggesting thatCRC-IV-1 channels also reside in the ER. This finding wasendorsed by costaining with a polyclonal mouse Ab (PDI; forAB characterization, see Fig. S3 in the supplemental material),which was raised against a recombinant peptide correspondingto Paramecium PDI 1-1 (PDI1-1; accession no. CAD99202)residues L33-E145, and the CRC-IV-1 specific Ab R772. Asshown in Fig. 4, double labeling occurred in the ER network(Fig. 4C), while R772 Abs solely stain scale-like structures in

the cell cortex (Fig. 4A and B). According to their position atthe cell periphery, shape, size, and arrangement, these struc-tures correspond to alveolar sacs. A detailed analysis by im-munogold electron microscopy indicates not only that CRC-IV-1 channels are present in these cortical Ca2� stores, butfurthermore that labeling is restricted to membranes at theouter side of alveolar sacs facing the cell membrane so that anyCa2� release would be spilled over trichocyst exocytotic sites(Fig. 5).

Gene silencing by feeding. Knockdown in Paramecium canbe achieved by feeding transformed bacteria which produce

FIG. 4. Colocalization studies using Abs against CRC-IV-1 and the ER resident PDI. Confocal images of a cell stained with purified polyclonalAbs against CRC-IV-1 (R772; green channel) and PDI (red channel). (A) Staining of the alveolar sacs exclusively by R772 Abs is shown by confocalslices taken from the cell surface. (B) Details are evident in an enlarged image. (C) In a median section plane, colocalization of both Abs occursonly in the ER. oc, oral cavity; cp, cytoproct. Scale bars, 10 �m.

FIG. 5. Immunogold electron microscopic localization of CRC-IV-1. Immunogold labeling with CRC-IV-1-specific Abs R883 (arrows) occursselectively on the outer part of the alveolar sacs (as) facing the cell membrane (cm). t, docked trichocyst. Bar, 0.1 �m.


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double-stranded RNA homologous to sections of the gene ofinterest (30). To achieve a knockdown phenotype of wholereceptor families, tandem constructs representing both iso-forms of IP3RN and CRC-IV-1 gene families were cloned intothe pPD gene silencing vector (see Materials and Methods).The constructs were designated pPD-N1N2 and pPD-C1C2,respectively (Fig. 6A). As a negative control, the ORF of GFPderived from the pPXV-GFP vector (35, 35) was cloned (pPD-GFP), whereas the pPD-ND7 construct was used as a positivecontrol (88), which contains the ORF of the “non-discharge”gene nd7, resulting in impaired exocytosis of trichocysts.

In order to show that the feeding constructs pPD-N1N2 andpPD-C1C2 lead to a specific knockdown, silencing of the chan-nels was monitored by analyzing mRNA levels via real-timePCR. Transcript abundances were quantified by using the Ct

relative quantification method (55). An intron-harboring sec-

tion of the Paramecium act1-1 (accession no. AJ537442) wasused for data normalization and as an internal control forsignals originating from any possible contaminating DNAs dur-ing the amplification step. As shown in Fig. 6B, mRNA levelsof CRC-IV-1 channels are reduced in pPD-C1C2-treated cellswithout affecting expression of IP3RN receptors, whereas treat-ment of cells with the pPD-N1N2 construct leads to decreasingtranscript levels of IP3RN receptors. We observed only slightlyreduced CRC-IV-1 transcript levels in pPD-N1N2-treatedcells, while we can confirm that the feeding constructs pPD-N1N2 and pPD-C1C2 each lead to a specific knockdown oftheir respective target genes.

To confirm that differential RNA expression is paralleled byconcomitant protein concentrations in silenced cells, we per-formed immunofluorescence analyses. For comparison, stain-ing procedures and imaging processing of silenced cells andmock-treated cells were performed under identical conditions.As shown in Fig. 7A and B, a strong reduction of R772-labelingof almost all CRC-IV-1-silenced cells (pPD-C1C2) could beobserved compared to control cells (pPD-GFP), whereas la-beling with Abs against �-tubulin still persists at the same level.Notably, the reduced R772 staining is not correlated withstructural alterations or modified arrangements of alveolarsacs in CRC-IV-1-silenced cells. Similar downregulation couldbe obtained, with the respective Abs, for IP3RN receptors ingene-silencing experiments. Since these CRCs localize to theCVC (53), cells silenced in these receptors through treatmentwith the pPD-N1N2 construct display a reduced CVC labelingcompared to control cells (pPD-GFP) when stained withIP3RN-specific Abs (R866; Fig. 7C), while the CVC still couldbe stained with Abs against the V-type H�-ATPase (Fig. 7D).As a marginal result, we only observed extremely swollen am-pullae or, in some cases, more severe perturbation of theorganelle (Fig. 7D).

General effects of gene silencing on cell activity. In a firstapproach, we examined whether silencing of IP3RN or CRC-IV-1 receptor families has an effect on Paramecium cell divi-sion. In general, a slightly reduced division rate during the first24 h of silencing could be observed, probably due to a lag effectafter transfer from normal medium to feeding solution (86).Furthermore, Ca2� concentration in the culture medium af-fects division rates of silenced cells. When [Ca2�]o of theculture medium was adjusted to 100 �M, which equals stan-dard conditions (76), cell division was not influenced by thesilencing constructs (Fig. 8A). However, when [Ca2�]o wasdecreased to 1 �M, a significant reduction of division rates ofpPD-C1C2- or pPD-N1N2-treated cells occurred, while con-trol cells treated with pPD-GFP or pPD-ND7 constructs dividejust as under standard conditions. A further reduction of[Ca2�]o to �0.3 �M by elevating the EGTA concentration to1.5 mM prolongs division cycles even in control cells and, overlonger times, causes cell death in pPD-C1C2- or pPD-N1N2-treated cells within 24 h. Taken together, these experimentsreveal that IP3RN channels and, to a lesser degree, CRC-IV-1channels are essential for viability in media with low [Ca2�]o

for reasons to be analyzed in more detail.We aimed at a more clear distinction between the two CRC

types by the following experiments. Considering the localiza-tion of IP3RN receptors to the CVC, we examined in whichknockdown situation this organelle would be affected. As

FIG. 6. Silencing of CRC-IV-1 and IP3RN genes by feeding.(A) Scheme of the silencing constructs for both isoforms of CRC-IV-1and IP3RN gene families represented by pPD-C1C2 and pPD-N1N2,respectively. Length and positions of the cloned fragments refer tocDNA sequences of each CRC. As a negative control, we applied theORF of GFP cloned in the pPD vector (pPD-GFP). As a positivecontrol, the pPD-ND7 construct was used, which contains the ORF ofthe nd7 gene impairing trichocyst exocytosis. C1 and C2 or N1 and N2,highlighted in red, indicate the positions of CRC-IV-1a (C1), CRC-IV-1b (C2), IP3RN-1 (N1) and IP3RN-2 (N2) specific amplicons usedfor real-time RT-PCR. (B) Expression pattern of CRC-IV-1 andIP3RN receptors during gene silencing. Total RNA (0.5 �g) was iso-lated from �4,000 Paramecium cells mock silenced with pPD-GFP,pPD-C1C2, and pPD-N1N2, each for 72 h. The relative abundances ofmRNAs of CRC-IV-1a (C1), CRC-IV-1b (C2), IP3RN-1 (N1), andIP3RN-2 (N2) were determined by real-time RT-PCR. The relativeexpression was calculated by using the ��CT method, and expressionlevels are given as the percentage of transcripts normalized to theact1-1 messenger. The data are means � the standard error of themean (SEM) from three replicate wells.


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shown in Fig. 8B, the filling and expelling cycle is clearly pro-longed from a value of �10 s in control cells to 16 s inIP3RN-silenced cells. In parallel, in some of these cells theCVC is severely perturbed as manifested by swollen ampullae

(Fig. 8C). In contrast, pPD-C1C2-treated cells do not showsuch alterations. The contractile vacuole shows regular, appar-ently even slightly shortened contraction periods (Fig. 8B), andno morphological anomaly could be detected (Fig. 8D).

FIG. 7. Immunolabeling of pPD-C1C2- and pPD-N1N2-treated cells. Confocal images of a control cell (pPD-GFP) (A) and a CRC-IV-1-silenced cell (pPD-C1C2) (B) using Abs against CRC-IV-1 (R772; green channel) and mouse monoclonal Abs against �-tubulin (DM1A; redchannel). CRC-IV-1 labeling of pPD-C1C2-treated cells is reduced after 72 h compared to control cells, whereas the labeling of basal bodies withAbs against �-tubulin (see enlargements) is not affected. The general arrangement of alveolar sacs and basal bodies is not altered in CRC-IV-1-silenced cells compared to control cells. Note the identical recording conditions for controls and 72-h silencing. (C) Immunofluorescence analysisof IP3RN1/2-silenced cells (pPD-N1N2) shows a reduction in labeling of the CVC with IP3RN-specific Abs (R866) compared to control cells(pPD-GFP). (D) Staining of pPD-N1N2-silenced cells with Abs against V-type H�-ATPase (V-ATPase [subunit a1-1]) persists, while some slightstructural alterations occur (of which several examples are presented). Most of pPD-N1N2-treated cells show expansions of the ampullae (arrows)and in some cells (upper middle image) the structure of the CVC appears to be less distinct than normally. Scale bars, 10 �m.


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Knockdown of IP3RN and CRC-IV-1 receptors affect exocy-tosis of trichocysts. Stimulation of trichocyst exocytosis leadsto rapid expansion of the secretory organelle contents in in-soluble needles. The exocytotic capacity of Paramecium cellscan be assayed by application of picric acid (79), which acts asa fixative allowing easy quantification of the amount of ex-truded trichocysts (Fig. 9A).

After 24 to 48 h of treatment with feeding bacteria, bothIP3RN- and CRC-IV-1-silenced cells show a significantly re-duced number of dischargeable trichocysts compared to con-trol cells. In contrast to pPD-ND7-treated cells, which displayan almost complete inhibition of exocytosis, silencing of thetwo CRC types analyzed leads only to a partial inhibition,individual cells showing different degrees of exocytosis reduc-tion (Fig. 9). Partial inhibition in pPD-C1C2-treated cells ismanifested by restricted extrusions of trichocysts either in clus-ters along the entire cell body or restricted to the cell poles. Incontrast, in IP3RN-silenced cells upon stimulation a restrictednumber of trichocysts is extruded over the whole-cell body,

also clearly in reduced amounts (Fig. 9A). Under these condi-tions we showed the ability of trichocyst contents to decon-dense, when Ca2� gets access (data not shown).

To visualize more clearly the effects of gene silencing onexocytotic capacity, we used Abs to stain TMP4, a componentof the inner trichocyst “core” region (98). Immunofluores-cence shows that the amount and the arrangement of tricho-cysts in pPD-C1C2-treated cells is comparable to that observedin control cells (pPD-GFP and pPD-ND7), where trichocystsare docked at the cell surface in high density (Fig. 10A). Incontrast, IP3RN-silenced cells display a reduced number oftrichocysts, which are mostly not attached at the surface. Sim-ilar results could be obtained with Abs against TMP1, whichrecognize the second mature polypeptide of TMP1b andthereby decorate the trichocyst “cortex” (98) (see Fig. S4A inthe supplemental material). This indicates that the generalshape of trichocysts is not altered in pPD-N1N2-treated cells.To confirm the results of the immunofluorescence studies,anti-TMP1 (see Fig. S4B in the supplemental material) and

FIG. 8. Additional aspects of gene silencing. (A) Influence of [Ca2�]o on IP3RN- and CRC-IV-1-silenced cells. Division rates of silenced cellswere determined by calculating the number of cell fissions per day. Black and dark gray bars represent the division rates of control cells (pPD-GFPand pPD-ND7) and light gray and white bars represent the division rates of CRC-IV-1-silenced (pPD-C1C2) and IP3RN-silenced cells (pPD-N1N2), respectively. The viability of cells treated either with pPD-C1C2 or pPD-N1N2 is reduced only when exposed to low [Ca2�]o compared tocontrol cells at the same [Ca2�]o: n (100 �M [Ca2�]o) � 40, n (1 �M [Ca2�]o) � 40, and n (�1 �M [Ca2�]o � 27. P values ([Ca2�]o �1 �M):C1C2 � 4.6181E09 and N1N2 � 9.84242E17. The P values correspond to the Student t test. Error bars indicate the SEM. (B) Pumping cyclesof the contractile vacuoles of IP3RN knockdown cells are prolonged (right bar) compared to control cells (pPD-GFP; left bar; P � 0.0003).Pumping cycles of CRC-VI-1 silenced cells are slightly shortened (pPD-C1C2; middle bar) with weak significance (P � 0.01 for pPD-C1C2- versuspPD-GFP-treated cells). The data were collected in four different experiments from at least seven cells per group: n (pPD-GFP) � 51, n(pPD-C1C2) � 30, and n (pPD-N1N2) � 43. Error bars indicate the SEM. (C) Representative of a live IP3RN-silenced cell showing extremelyexpanded ampullae (white arrows) attached to the contractile vacuole. See also the enlargement in panel C�. A further conspicuous detail is theswollen cell body combined with accumulations of large crystalline inclusions in the cytosol (black arrows). (D) Representative of a liveCRC-IV-1-silenced cell. In contrast to IP3RN-silenced cells, the contractile vacuole is not affected. See also the enlargement in panel D�. Scale bars,10 �m.


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anti-TMP4 Abs (Fig. 10B and C) were also used in Westernblots. As shown in Fig. 10B, the intensity of the band repre-senting the mature TMP4 polypeptide of �18 kDa is clearlyreduced in preparations from IP3RN silenced cells comparedto extracts from pPD-GFP-, pPD-ND7-, and pPD-C1C2-treated cells. This indicates that the reduction of exocytoticcapacity of IP3RN-silenced cells is due to decreasing amountsof trichocysts in these cells, suggesting that the biogenesis oftrichocysts is affected due to altered Ca2� homeostasis. CRC-IV-1-silenced cells also displayed reduced exocytosis but, incontrast to IP3RN-silenced cells, the amount and the arrange-ment of trichocysts is not affected. This supports an involve-ment of CRC-IV-1 channels in signal transduction.

Influence of [Ca2�]o on trichocyst biogenesis. We hypothe-sized that the reduced trichocyst content in IP3RN-silencedcells is a secondary effect due to altered [Ca2�]i, as IP3RN

receptors are supposed to be involved in graded reflux of Ca2�

to fine-tune [Ca2�]i homeostasis (53). Therefore, we investi-

gated a possible correlation of trichocyst biogenesis and[Ca2�]i, by incubating cells in media with decreasing [Ca2�]o,to which intracellular concentrations, although at a much lowerlevel, rapidly adjust in these cells (12, 24, 43). Viability at low[Ca2�]o is demonstrated in Fig. 11A, showing also that divisioncycles are accelerated with rising [Ca2�]o. By stepwise lowering[Ca2�]o levels, the number of discharged trichocysts decreasesgradually (Fig. 11B). To investigate whether the reduced exo-cytotic capacity results from a reduced number of trichocysts,we prepared whole-cell homogenates and performed Westernblot analyses with Abs against TMP4. As shown in Fig. 10C,the amount of TMP4 decreases when cells are grown in mediawhere [Ca2�]o was reduced to a threshold of tolerable low[Ca2�]o. This suggests that the reduced number of trichocystsin IP3RN-silenced cells might be a secondary effect of lowered[Ca2�]i due to impaired Ca2� reflux as a consequence of low-ered IP3RN-type receptor levels.

Silencing of CRC-IV-1 channels results in impaired Ca2�

release from cortical stores. Are Ca2� transients during stim-ulated exocytosis reduced in CRC-IV-1-silenced cells? FuraRed-loaded cells were triggered with AED and changes in[Ca2�]i were monitored by double-wavelength recordings. Asthe source of Ca2� during stimulation includes not only Ca2�

mobilization from the alveolar sacs but also a superimposedCa2� influx from the medium (46, 72), we performed theexperiments at a [Ca2�]o slightly below [Ca2�]i in unstimulatedcells (see Materials and Methods) to visualize Ca2� transientssolely emerging from alveolar sacs (Fig. 12A). Clearly, CRC-IV-1 silencing results in a significant reduction of Ca2� releas-able from stores compared to controls (pPD-GFP). Sinceformer studies had shown that the ryanodine receptor activa-tors, 4-CmC and caffeine, activate Ca2� release from alveolarsacs (46, 48), we also applied these drugs to mock-treated andto CRC-IV-1-silenced cells. As shown in Fig. 12A, stimulationof cells with either 4-CmC or caffeine results in similar Ca2�

transients as observed with AED. Under these conditions, tri-chocyst exocytosis is almost totally inhibited. Since this, how-ever, could theoretically also be due to the requirement ofextracellular Ca2� for the decondensation and expulsion oftrichocyst contents (10), we repeated the experiments withCRC-IV-1 silenced cells in the presence of standard [Ca2�]o

(Fig. 12B). We found that upon stimulation with the threecompounds the [Ca2�]i signals generated are diminished (Fig.12B), and the number of discharged trichocysts in that cases isalso greatly reduced (to �10%) in comparison to values ob-tained with control cells (data not shown). This clearly indi-cates that CRC-IV-1 silencing bears on the Ca2� signal re-quired for exocytotic membrane fusion.

DISCUSSION

Although a plethora of plasmalemmal cation channels arelargely characterized in Paramecium (51, 58), identification ofintracellular CRCs has remained elusive. Since the Parame-cium genome database has become developed (5, 21), westarted identifying CRCs. As outlined in Results, these maycontain some unexpected combinations of domain sequences,recalling in part IP3 or ryanodine receptors, while large partsare unrelated to any known channel structures. Thus far, wehave compared the size, the presence of IP3 binding and the

FIG. 9. Knockdown of both CRC types results in reduced tricho-cyst exocytosis. (A) Bright-field images show representatives of si-lenced cells treated with picric acid to mimic exocytosis (see Materialsand Methods). Trichocysts elongate upon expulsion and become visi-ble as needles (several times longer than cilia) attached to the cells.Both cells silenced in CRC-IV-1 (pPD-C1C2) and in IP3RN (pPD-N1N2), respectively, show reduced exocytosis of trichocysts comparedto control cells (pPD-GFP). Silencing of the ND7 gene (pPD-ND7),our positive control, leads to almost complete inhibition of trichocystextrusion. Scale bar, 10 �m. (B) Quantitation of exocytotic capacityafter 48 h of feeding tested with picric acid. The graph shows quanti-tative distributions of silenced cells with different degrees of exocytosisinhibition. The data were collected in seven experiments. Error barsindicate the SEM. n (pPD-GFP) � 262; n (pPD-ND7) � 266; n(pPD-C1C2) � 266; n (pPD-N1N2) � 241.


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RIH domain structure, putative transmembrane regions, andthe nature of the pore domain, as derived from gene structure,but functional analyses have been restricted to CRC-II-1(IP3Rs) (53) and CRC-IV-1 channels (the present study).Thus, the channels not addressed in the present study are to beconsidered only bona fide Ca2� channels whose properties stillhave to be established. Subfamily CRC-IV-1 is a novel channeltype. Since both CRC types are involved in regulating distinctsteps of the secretory cycle in Paramecium, we now comparethese two groups of CRCs.

Effects of CRC-II-1 silencing. These experiments aimed atestablishing specific effects of the two P. tetraurelia CRCs un-der consideration here. Silencing of CRC-II-1 channels causesreduced numbers of trichocysts that mostly stay off the cellmembrane, while after silencing of CRC-IV-1 a full set oftrichocysts remains attached to the cell membrane in the ab-sence of exocytotic capacity. One may take into account dif-ferent Ca2�-based effects, particularly since CRC-II-1 chan-nels are known to deal with the regulation of basal [Ca2�]i

homeostasis (53). Lack of Ca2� reflux from the CVC, whichthey were shown to serve, could compromise several subcellu-

lar activities. Indeed, our experiments with control cells at low[Ca2�]o also result in a reduced number of trichocysts.

Theoretically a primary effect of interfering with Ca2� ho-meostasis by CRC-II-1 silencing could be the requirement of aCa2�-activated calreticulin complex for correct processing andfolding of secretory proteins (20, 38). Although a calreticulin-like protein occurs in Paramecium (73), we see in Westernblots, after CRC-II-1 silencing, a correctly cleaved TMP4 pro-tein comparable to the size of processed TMPs in normal cells(31, 99). The small number of trichocysts, mostly remainingunattached inside the cytoplasm, observed after CRC-II-1 si-lencing may largely represent residual organelles persistingfrom before gene silencing. This may be explained as follows.

A salient aspect concerning reduced trichocyst numbers af-ter CRC-II-1 silencing may concern budding of precursor ves-icles and their transport and fusion, as this also occurs duringtrichocyst biogenesis (99). In higher eukaryotic cells, some ofthese processes, or steps thereof, are known to be subject toCa2� regulation (1, 6). In P. tetraurelia, all steps of exo- andendocytosis are known to be accelerated by increased [Ca2�](72), whereas no information is available on trichocyst biogen-

FIG. 10. Knockdown of IP3RN, but not CRC-IV-1, leads to decreasing amounts of trichocysts. (A) Confocal images of silenced cells stainedwith Abs against TMP4. Representatives of control cells (pPD-GFP and pPD-ND7) or CRC-IV-1-silenced cells (pPD-C1C2) show trichocystsdocked in high density at the cell surface. In pPD-N1N2-treated cells, the overall number of trichocysts and their docking at the cell surface isreduced. Stacks were fused from 20 (pPD-ND7, pPD-C1C2, and pPD-N1N2) or 13 (pPD-GFP) confocal slices (0.75-�m thickness). Enlargementsshow median sections to visualize the arrangement of trichocysts at the cell periphery. Scale bars, 10 �m. (B) Western blot of protein preparationsfrom silenced cells treated for 72 h with either pPD-GFP, pPD-ND7, pPD-C1C2, or pPD-N1N2. As a loading control, the same blot was probedwith Abs against �-tubulin (�-tub, upper panel) showing similar protein amounts in each lane. In extracts prepared from pPD-N1N2-treated cells,Abs against TMP4 (lower panel) recognize a significantly reduced amount of TMP4 in pPD-N1N2-treated cells. (C) Quantitation of gray valuesof TMP4 signals in Western blots substantiates reduced TMP4 contents in pPD-N1N2-treated cells compared to control cells. There is only a slightincrease of TMP4 signal in pPD-ND7, and a slight decrease (also not significant) in pPD-C1C2-treated cells in comparison to controls.


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esis. In contrast, in higher eukaryotes intracellular transport ofnewly formed secretory organelles has been described as beinginsensitive to [Ca2�] (68). From this one may conclude that thereduced number of total trichocysts may be mainly due toinhibited biogenesis which, with ongoing division activity dur-

ing CRC-II silencing, also entails a reduced number of docked/dischargeable trichocysts.

In Paramecium, just as in other secretory systems, a cytosolicCa2� signal is required for membrane fusion (24, 47), whileexogenous Ca2� is required for contents release (10). Dense

FIG. 11. Biogenesis of trichocysts depends on [Ca2�]o. (A) Division rates of cells grown in media supplemented with EGTA and CaCl2 weredetermined by calculating the number of cell fissions per day. After 48 h of Ca2�/EGTA treatment, division rates of cells decrease with lowering[Ca2�]o (P � 0.036 for 1 mM EGTA–0.8 mM CaCl2 versus 1 mM EGTA–1 mM CaCl2; P � 0.017 for 1 mM EGTA–0.6 mM CaCl2 versus 1 mMEGTA–1 mM CaCl2; P � 0.0001 for 1 mM EGTA–0.4 mM CaCl2 versus 1 mM EGTA–1 mM CaCl2; P values according to the Student t test).Error bars indicate the SEM. n (aliquots of counted cells) � 8. (B) Exocytotic capacity of cells grown for 48 h in media with different [Ca2�]o wasquantified by using the picric acid test. Note the increasing exocytosis with increasing [Ca2�]o. n (1 mM EGTA–1 mM CaCl2) � 52; n (1 mMEGTA–0.8 mM CaCl2) � 45; n (1 mM EGTA–0.6 mM CaCl2) � 48; n (1 mM EGTA–0.4 mM CaCl2) � 57. (C) Western blot analysis ofhomogenates of cells incubated with different [Ca2�]o for 48 h and probed with anti-TMP4 Abs. The amount of TMP4 diminishes when cells weregrown in media with reduced [Ca2�]o levels. As a loading control 12.5 �g of the protein samples were analyzed by SDS-PAGE, followed by silverstaining according to the method of Wray et al. (105).

FIG. 12. Knockdown of CRC-IV-1 channels impairs Ca2� release from cortical stores. Time course of [Ca2�]i changes in cortical regions ofFura Red-injected cells, silenced in CRC-IV-1 receptors (red), or mock-treated cells (pPD-GFP, black), following stimulation with AED or theryanodine receptor agonists, 4-CmC and caffeine. Stimulation was performed at low [Ca2�]o by adding 1 mM BAPTA to yield [Ca2�]o � 30 nM(A) or under standard conditions ([Ca2�]o � 100 �M) (B).


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core-secretory vesicles in metazoans usually contain a ratherhigh calcium content, e.g., tens of millimolar (15, 67), butorganellar Ca2� is not detectable in trichocysts by X-ray mi-croanalysis, in contrast to that in alveolar sacs of Paramecium(33). Only access of extracellular Ca2� to trichocyst contentsvia the fusion pore can cause their “decondensation” (sever-alfold stretching upon vigorous exocytotic discharge) (10). Thiscapability is retained by the residual trichocysts remaining afterCRC-II silencing, while de novo formation is inhibited.

In essence, the Ca2�-based phenomena outlined above canexplain reduced trichocyst biogenesis and consequently re-duced docking after CRC-II-1 silencing. All of these mecha-nisms await scrutinized investigation. Another salient point ofour work, however, is that both families of P. tetraurelia CRCsanalyzed exert widely different functions in the secretory cycleof Paramecium.

Functional assignment of CRC-IV-1 channels to alveolarsacs. We currently find CRC-IV-1 immunolabeling on alveolarsacs and in the ER. How does that compare to the previousfinding that the Ca2� signals required for stimulated exocytosiswould originate primarily from alveolar sacs (75)? Clearly, theconsiderable identity (nearly 85%) of the ORFs of both CRC-IV-1 genes would entail that silencing would affect both iso-forms, since differential downregulation would require 15%difference in identity (85). Close similarity also restrains usfrom any differential Ab labeling, whereas expression as GFPfusion proteins is hampered by the mere size of the CRCmolecules.

Bearing in mind the considerable ambiguity of pharmaco-logical tools in work with ciliated protozoans (77), we ratherreanalyzed the effects of different trigger agents previouslyused, among them polyamines (49, 74, 78) and the permeableRyR agonists, 4-CmC and caffeine, which all normally producetrichocyst exocytosis in parallel to SOC-type Ca2� signaling(46–48).

We now observe by fluorochrome analysis after CRC-IV-1silencing that, with all of three trigger agents, exocytosis isinhibited in parallel to a reduction of the Ca2� release fromalveolar sacs. In conjunction with the subcellular localizationof CRC-IV-1 to alveolar sacs, the generation of Ca2� signals,therefore, supports the assumption that CRC-IV-1 channelsare the Ca2�-release channels relevant for trichocyst exocy-tosis.

Despite the insensitivity of CRCs in alveolar sacs, now iden-tified as CRC-IV-1 channels, to ryanodine (54), their sensitivityto 4-CmC (46) and caffeine (48) clearly argues for RyR-relatedchannels, since this is characteristic of RyRs in higher eukary-otic systems (22, 26, 39). Unfortunately, no binding domainsare known for caffeine, which also requires tens of millimolarconcentrations in other cells, just as in Paramecium (25, 48).Putative ryanodine-binding sites normally present in the lastC-terminal transmembrane domain (100) are not present inthe primary sequence of P. tetraurelia CRC-IV-1 (Fig. 2D),thus explaining their insensitivity. 4-CmC binding sites in RyRsare also well characterized in higher eukaryotes (26, 27). Com-parison with Paramecium sequences reveals that this site isconserved in CRC-IV-1 channels (see Fig. S2 in the supple-mental material). Their insensitivity to ryanodine, therefore,recalls the situation in malignant hyperthermia patients whosemutated RyRs are sensitive selectively to 4-CmC (102). Our

data are also supported by electrophysiological recordingsfrom lipid-reconstituted P. tetraurelia cortex fragments (108)whereby several channel functions have been detected, one ofthem with properties of an organellar Ca2�-release channel ofthe type we describe here.

Diversification of Paramecium CRCs. The presence of 34proteins distantly related to IP3Rs and RyRs in the Parame-cium genome reflects a wide expansion of such channels com-pared to the situation in metazoans (see “Indroduction”) and,as known thus far, in other unicellular systems (4, 13, 96) (Fig.1 and 13). A similarly broad diversification has been observedalso within many other Paramecium gene families (32, 84, 86,101), which can be explained as follows. Owing to a recentwhole-genome duplication, Paramecium genes often occur aspairs of closely related paralogues (5), which is the case with 20genes of the 34 CRCs described here. Expansions within genefamilies in Paramecium may compensate for the absence ofalternative mRNA splicing (40), a pathway known to ensurediversification of mammalian IP3Rs and RyRs (9, 28). Weassume a similar situation for Tetrahymena, which according tothe TransportDB (http://www.membranetransport.org [83])possesses 23 CRCs. Thus, expansion of such channel typesseems to be a significant feature of ciliates.

The Paramecium channels we describe as bona fide CRCs(Fig. 1) represent novel types, with the exception of the re-cently described CRC-II-1 channels, which possess typical fea-tures of IP3Rs (53). The other P. tetraurelia CRCs displayunusual combinations of one or several characteristics ofIP3Rs, of RyRs, of both, or of unrelated parts. Figure 1Apresents the likely relationship between the P. tetraureliaCRCs, based on ORF analysis. (Note that RyRs have not beenincluded in Fig. 1A because, due to their size, only conservedregions of the channel domains are considered in alignments,which would lead to cluster formation with mammalian IP3Rs.)It appears that precursor channels would have undergone in-creasing diversification to serve the multiple specialized func-tions of Ca2� signals in Paramecium (see the introduction). Incontrast to the most stringently defined CRC-II-1 channels(53), CRC-IV-1 channels display some aberrant features, asoutlined in “molecular characterization of CRC-IV-1 chan-nels.” The other subfamilies remain to be characterized bydetailed domain analysis and functional assays, a topic forfuture work.

Evolutionary aspects. Currently, Ca2� signaling in unicellu-lar eukaryotes meets considerable interest. In the closely re-lated parasites Plasmodium and Toxoplasma (both being mem-bers of the phylum Alveolata, just like ciliates), Ca2� signalingis mandatory for host cell penetration (17). However, it is notyet known whether the “inner membrane complexes” (alveolarsac-like structures) play a role in Ca2� signaling (16). IP3Rsare assumed to occur, although other types—yet to be identi-fied—have also been envisaged (57, 65). In sum, clear molec-ular identification of CRCs in these organisms has not beenpossible thus far, despite considerable efforts (66). A similarsituation occurs in the unicellular fungi, Candida albicans andNeurospora crassa. For both species IP3-dependent Ca2� sig-naling is evident (14, 87) (see the introduction), but no IP3Rorthologues show up in genomic screens (note that for all ofthese organisms complete genome information exists). Thissuggests that evolution has led to diversification to an extend


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that the perceptibility of IP3R-related sequences may be re-stricted to a limited number of unicellular species.

According to the TransportDB (83), which allows genome-wide comparison of predicted membrane transporters across abroad range of organisms, IP3R-related genes are present inseveral unicellular organisms (Fig. 13). In addition, to ciliateCRCs, they exist in the algae Chlamydomonas reinhardtii andAureococcus anophagefferens (note that they are absent fromhigher plants [103, 106]) and in several Phytophthora species.Among protozoan parasites, IP3R-related genes occur inTrypanosoma and Leishmania species. There exist four genescoding for IP3Rs (13) in the choanoflagellate Monosiga brevi-collis, which were identified in a database screen of the recentlyunraveled genome (http://genome.jgi-psf.org/Monbr1 [44]), aswell as the IP3, receptor-like protein IplA (96) from Dictyoste-lium discoideum.

In fact, such sequences diverge from metazoan IP3Rs to asimilar degree as observed for Paramecium CRCs (Fig. 13).Alignments with Mus musculus IP3R1 reveal, that conserved

parts of orthologues from protozoa are mainly pronounced inregions of the C-terminal channel domain (Fig. 13B; for de-tails, see Fig. S5 and Table S4 in the supplemental material).An exceptional position holds true for CRCs from M. brevicol-lis (13) with clearly recognizable similarities to both, ciliateCRCs and mammalian IP3Rs (Fig. 13). The recent identifica-tion of such channels in M. brevicollis, although not yet ana-lyzed in any detail, suggests an IP3R-mediated mechanism inchoanoflagellates (13). Their comparatively high degree ofconservation correlates with the evolutionary position of theseorganisms as putative precursors of metazoans (44, 60). Con-comitantly, recent genomic analysis of the M. brevicollis ge-nome (44) has suggested that complex Ca2� signaling mayhave evolved early on (13).

The IP3 type of signal transduction is clearly present alreadyin Paramecium (53). Beyond that, we now find stringent evi-dence of additional CRCs, including the novel CRC-IV-1channels. These may belong to an evolutionarily old type ofsignal transduction pathway. Taken the number of subfamilies

FIG. 13. IP3R-related protozoan sequences. (A) Neighbor-joining tree (with 1,000 bootstrap replicates) of phylogenetic relationships betweenIP3R-related orthologues. Note that systematic groups are designated by (a to k). For deuterostomes (a and b), we included sequences representingIP3Rs from the mammalian M. musculus (MmIP3R1) (a) and from the echinoderm Asterina pectinifera (b). For protostomes, we included IP3Rsfrom D. melanogaster (c) and from the nematodes C. elegans and Brugia malayi (d). Among unicellular organisms orthologues are present in thechoanoflagellate Monosiga brevicollis (e) and in the ciliates P. tetraurelia and T. thermophila (f). IP3R-related genes also exist in Phytophthorainfestans (i), the green algae Chlamydomonas reinhardtii (j), the chrysophycean algae Aureococcus anophagefferens (k). and the iplA gene ofDictyostelium discoideum (g) (96). (h) Among protozoan parasites, IP3R orthologues are present in trypanosomes such as Leishmania major,Leishmania infantum, Trypanosoma brucei, and Trypanosoma cruzi. Sequences were identified either by BLASTP searches of respective databasesor by using the TransportDB (http://www.membranetransport.org [83]). Accession numbers of the sequences are summarized in Table S4 in thesupplemental material. Bootstrap support values for the nodes are indicated, as are evolutionary distances by the scale bar, below. (B) Sequenceanalysis of IP3R-related protozoan sequences. Conserved regions were determined by using the BLASTP 2.2.19 program (http://www.ncbi.nlm.nih.gov [3]) with the same proteins as those presented in panel A, aligned with M. musculus IP3R1 as query sequence. Alignments are color codedby score with five score ranges. The positions of flanking residues and corresponding e-values are summarized in Table S4 in the supplementalmaterial.


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of P. tetraurelia CRCs together, CRCs may have diversifiedearly on in evolution to an unexpected extent, involving notonly IP3Rs and RyR types, but additional ones still to becharacterized in detail.

ACKNOWLEDGMENTS

We thank Jean Cohen and Linda Sperling (CNRS, Gif-Sur-Yvette,France) for enabling us to perform the screening experiments andadditionally Genoscope (Evry, France) for access to the server with theParamecium sequencing data. We thank especially Linda Sperling forproviding the Abs to TMP1 and TMP4, as well as for help in organizingaccession numbers from the Paramecium database. We are grateful toMarek Zagulsky (Polish Academy of Science, Warsaw, Poland) forinitial help with sequencing at an early stage of this work. We alsothank Thomas Wassmer and Roland Kissmehl for helpful discussions,Deisy Geisinger for providing the PDI expression plasmid, and Lau-retta Nejedli for technical assistance (all at the University of Con-stance, Constance, Germany). In addition, we thank E. May for use ofthe LSM510 facilities and A. Burkle for access to the Bio-Rad iCycler.

This study has been supported by the Deutsche Forschungsgemein-schaft (grants to H.P., including project C4 of TR-SFB11).

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