2 Novel Types of Ca + Release Channels Participate In the Secretory Cycle of Paramecium Cells Eva-Maria Ladenburger,* Ivonne M. Sehring, Iris Korn, and Helmut Plattner Department 0/ Biology, University of Cons/ance, 78457 Constance, Germany

A database search of the Paramecium genome reveals 34 genes related to Ca2+ -release channels of the inositol-l,4,S-trisphosphate (IP) or ryanodine receptor type (IP3 R, RyR). Phylogenetic analyses show that these Ca2+ release channels (CRCs) can be subdivided into six groups (Paramecium tetraurelia CRC-I to CRC-VI), each one with features in part reminiscent of 1P3Rs and RyRs. We characterize here the P. tetraurelia CRC-IV-l gene family, whose relationship to IP.,Rs and RyRs is restricted to their C-terminal channel domain. CRC-IV-l channels localize to cortical Ca2+ stores (alveolar sacs) and also to the

endoplasmic reticulum. This is in contrast to a recently described true IP3 channel, a group II member (P. tetrallrelia IP3RN -l), found associated with the contractile vacuole system. Silencing of either one of these CRCs results in reduced exocytosis of dense core vesicles (trichocysts), although for different reasons.

Knockdown of P. tetraurelia IP3 R N affects trichocyst biogenesis, while CRC-IV-l channels are involved in signal transduction since silenced cells show an impaired release of Ca2+ from cortical stores in response to exocytotic stimuli. Our discovery of a range of CRCs in Paramecium indicates that protozoans already have evolved multiple ways for the use of Ca2+ as signaling molecule.

2 z Ca + is an important component of cell activity in all organ­ action potentials involving increased [Ca +]; (93). Treatment

isms, from protozoa to mammals. Thereby Ca2+ may originate of vacuolar membrane vesicles from Candida albicans with IP3 from the outside medium and/or from internal stores (7, 18). results in Ca2+ release, blocked by heparin and ruthenium red 2 2 + 2 Ca release from internal stores is mediated by various Ca + (14). IP3 generates and maintains a Ca + gradient in the hy­ release channels (CRCs), of which the inositol-1,4,5-tri sphos­ phal tip of Neurospora crassa and the IP3-sensitive channels phate receptor (IP3R) and ryanodine receptor (RyR) families have been reconstituted and characterized with the planar have been studied most extensively (8, 9, 29, 63). IP3 Rs and bilayer method (87). In summary, these publications suggest RyRs have been identifi ed in various metazoan organisms (re­ that IP3-dependent signaling pathways are conserved among viewed in references 9, 28, and 104). According to these re­ unicellular organisms, including protozoa. views, there exist three genetically distinct isoforms of each Despite these data, the molecular characterization of IP) or receptor type in mammals and orthologues have been identi­ ryanodine receptors in low eukaryotes is currently a challenge fied in various nonm alllmal ian vertebrates, e.g., frogs, chi ck­ sinl:l: thl: itil:nti(jl:ation or orthologul:s has not bccn possible ens, and (-ish. RyRs and JP3Rs were also cloned and sequenced thus far, probably because of evolutionary sequence diver­ in the invertebrates Drosophila melanogaster and Caenorhabdi­ gence (66). Traynor et al. (96) identified an IP3 receptor-like tis elegans, which possess one copy of each receptor type. protein, IpIA, in Dictyostelium discoideum, which possesses re­ Functional evid ence for Ca2 + release in response to ryano­ gions related to IP3R sequences, but thus far no evidence for dine or IP, receptor agonists has been described in several IP, interaction exists. We have recently described an IP,R in unicellular systems. Treatment of permeabilized Plasmodium the ciliated protozoa Paramecium tetraurelia (referred to here H chabaudi parasites with IP3 results in Ca release, which is as P. tetraurelia IP3R N ) (53), with features characteristic of inhibited by the IP3 receptor antagonist heparin (69). Another mammalian IP JRs in terms of topology and ability for IP, apicomplexan parasite, Toxoplasma gondii, responds to ago­ binding. The expression level of P. tetraurelia IP3R N is modu­ nists and antagonists of both, ryanodine and IP, receptors, by lated by extracellular Caz-!- concentrations ([Ca"+lu) and im­ mediating increases in intracellular Ca2 + concentration 2 munofluorescence studies reveal an un ex pected locali za ti on to ([Ca " ];) (56). Stimulation of T,ypanosoma cruzi with carba­ 2 the contractile vacuole complex (CYC), the major organelle chol results in increased [Ca ' ]; and IP) (59). IP, and cyclic involved in osmoregulation (2). The ionic composition of the ADP-ribose induces Caz, release in Euglena gracilis micro­ cont ractile vacuole fluid by ion-selective microelectrodes (91) some fractions in a dose-dependent manner (61). In the giant suggests that the organelle plays a major role in expelling an algae Cham corallina and Nitrella translucens, IP) produces 2 excess of cytosolic Ca ·'· . Therefore, these IP3 Rs may here mediate a latent, graded reflux of CaH for fine-tuning of 2 2 * Corresponding author. Mailing address: Department of Biol­ [Ca , ]; and thus serve [Ca ' ] homeostasis (53). ogy, University of Constance, P.O. Box 5560, 78457 Constance, Besides [Caz-!- ] homeostasis, the Paramecium cell has to Germany. Phone: 49-7531 -884230. Fax: 49-7531-882245. E-mail: regulate a variety of we ll -characterized processes (75). This [email protected]. includes exocytosis of dense-core secretory vesicles (tricho­ cysts) (7.1, 74, 99). Each cell possesses up to 1,000 trichocysts attached to the cell membrane. Their contents can be extruded

3605 3606

synchronously in response to natural stimuli, i. e., predators sec Table S I in the supplcmental material). PC Rs were carried out in 30 cycles (34, as co nfirmed by Knoll el al. [49]), to artificial polyamine of 95°C for 30 s, 54°C for 30 s, and 68°C for '1 2 min. The complete open reading fram c (ORF) ofCRC-IV- la and parts of th c ORF ofCRC-TV- 1h wcre identified secretagogues such as aminoethyldextran (AED) (78), to caf­ by amplifyi ng mRNA sequences by revcrse transcription-PCR (RT-PCR). Prep­ feine (48) 0 1' to the ryanodine substitute, 4-chloro-meta-cresol aration of RNA and cDNA synthesis was pcrformed as descri bed previously (53). (4-CmC) (46). Their expulsion strictly depends on Ca2 + (10) PCR amplifica ti on of cDNAs occulTed with thc Advanlage 2 PCR enzy me and is accompanied by an increase of intracellular [Ca2 +L (24, system (Clontech, Palo Al to, CAl (for primers, sec Table S1 in the supplcmental 47). T hi s Ca2+ signal origin ates from rapid mobilization of mate rial), and reactions were carried out in 35 cycles of 95°C fo r 30 s, 53°C for 2U s. and 68°C for 120 s. PCR-amplilied macronuciear DNA and cONA frag­ cortical stores, the alveolar sacs (33, 64, 74), superimposed by ments were cloned in the pCRII-TOPO clonin g system (Invit roge n, Carlsbad, Ca2+ influx (46, 72). It thus represents a SOC-type mechani sm CAl and analyzed by sequencing (Emolins MWG Operon. Marlinsried, Ger­ (SOC, store-operated Ca2 + entry) known from mammalian many). systems (81). Abs. To produce anti bodics (Abs) to CRC-IV- l , we amplifie d macronuclcar Upon exocytosis stimulation - 60% of their total Ca2+ is DNA corresponding to resid ues P2458 to I-l 2556 of CRC-IV-I a by PCR (for 2 primers, sec Table S I in the supplemental material) and cloned the fr agment into released from alveolar sacs (33). These are Ca + stores (90) th e pETIlih ex pression vector (Novagen. Madison. WI). Puriflcation of th e represented by fl at membrane compartm ents ti ghtly attached His-tagged P2458-H2556 peptide, as we ll as A I> producti on and puri fica ti on, was at the cell membrane surrounding each trichocyst docking site. performed as described previously (53). They possess a SERCA-type pump located at the membrane The a mnity purified Abs (R772 and R883) were used at coneenr rari ons of 12 ~glm l (R772) or 5 ~glm l (R883) in immunofluorescence studies and 0. 25 ~glm l facing the cell center (36, 37) and a luminal high-capacity/low­ in Western blots. T he polyciunal Ill uuse Ab tl irected against protein di sullide affi nity CaBP of the ca lsequestri n type (73). Thus far, Ca2+ isomerase (POI) fr om P. le/mllrelia was ra ised against a polypeptide correspond­ release ch.a nn els of th ese stores were identified only indirectly ing to N-terminal amino acids L33-E I45 of POII-1 (accession no. CAD99202). as cells respond by exocytosis to the RyR activators caffein e The serum obtained after repeated injecti ons of the antigen was directly used; (54, 48) and 4-CmC (46). However, an involvement of con­ eithe r di luted '1:1,000 for Western blots or 1:100 for immunostainings. Abs against trichocyst matrix protein I (TMP I) and trichocyst matrix prote in 4 served RyRs has remained questionable as ryanodine is not (TMP4) were generously provided by Linda Sperling (Centre de Gcnc tique 2 able to activate Ca + release from alveolar sacs, as is the case Molccul ai,.e, Centre National de la Reche rche Scientifique. Gif-sur-Yvelle.

with IP1 (54). Therefore, one of the most intriguing questions France) and used diluted 1:600 (TMPI) or 1:800 (TMP4) for immunostainings is the elucidation of the molecular nature of the channels and 1:4,000 (TMPI) or 1: 8,000 (TMP4) for Western blots, respectively. The Ab directed aga inst V -type 1-1+ -ATPase is described in Wass­ mediating Ca2+ release from alveolar sacs upon stimulated polyclonal rabbit mer et al. ( 101 ) (there designated as "anti-a'I- I" IP I78-S328J) and was used at a exocytosis. co ncentration of 12 ,"glml in immunostaini ngs. The Ab against a-tubulin was a In the present work we describe a novel family of CRCs (P. monocl onal mouse Ab (clone DMIA; Sigma- Aldrich, Schnelldorf, Germany), telraurelia CRC-lV-l), whose members display several proper­ whi ch was used diluted 1:200 in immunostainings and I :4,000 in Western blots. ti cs of th e chan nels postulated above. In detail, th e id entifi ed Cell harvesting, protein preparation, and Western blots. Cell s were harvested by centrifugati on (2 min; 100 X g), washcd twice with PIPES buffer (pH 7.0) CRC-IV-1 channels localize to th e alveolar sacs. Functional co ntai ni ng 5 mM PIPES (piperazine- l,4-bisI2-ethanesulfonic acid!), 1 mM KCI

and tluo rochro me analyses aIler gene sil encin g reveallhatlhey and I mM CaCI2 • Aliquots (100 ,"I) were frozen in li quid nitrogen. Samples we re are essential for mediating Ca2 + release and exocytosis in th awed in the presence of a protease in hi bitor cockt ail (complete; Roche Diag­ response to AED, ca ffe in e, or 4-CmC. Their cl assifi ca ti on as nostics, Basel, Switzerl and), 400 ~ I of methanol and 100 ~ I of chloroform. After "novel" CRC lype is based on a restricted relationship to the the addition of 300 ~ I of double-di stilled H20 probes were centrifuged for 5 min at 4,500 X g, and the aqueous phase was removed, fo ll owed by addin g 300 ,"I o f C-terminal channel domains of IP]Rs and RyRs. The overall methanol. After vortexin g and a second centrifugati on step (5 min, 4,500 x g), size and the number of putative transmembrane domains re­ the supern atant was removed, and the pe ll et was air dried. Pellets were dissolved semble lP}Rs, but N-terminal parts of CRC-lV-1 channels do in 2.5% sodium dodecyl sulfate (SOS) and complemented with tenfold-concen­ not show any conservation, such as an IP3-binding domain . trated sampl e buffe r (0. 1 M Tris- HCI IpH 81,50% (3-mereaptocthanol, 32% glycerol). Proteins (10 ,"gliane) were separated by SOS-polyacry lamide gel e1ec­ Therefore, CRC-lV-l channels represent distant relatives of trophorcsis (PAGE) accordin g to the method of Laemmli on 12% polyacrylam­ IP3Rs a nd RyRs and may belong to an ancestral Ca2+ signaling ide gels and transfe rred onto nitrocellulose membranes. Me mbrancs were pathway. blocked with 5% nonfat dry milk dissolved in Tris-buffc red sali ne plus Tween 20 ( 10 mM Tris-HCI, 150 mM NaCI, 0.05% Tween 20 IpH 8J) and incub ated for 1 h with pri mary Abs. Detecti on occurred by goat an ti -rabbit or goat anti-mouse MATERIALS AND METHODS immunoglobulin G (lgG) coupled to horseradish peroxidase (I :25,000; Dianova, I'arall/ecillll/ cultures. P. lelrallrelia wil d- type stocks 7S and d4-2 derivcd from Hamburg, Germany); luminescence was vis uali zed with an ECL Weste rn blolling stock 5 1S (89) wc re cultiva ted at 25°C in a decoeti on of dried lettuce (SM system (Amersham, Frciburg, Germany). med ium ) supplemented with 0.4 ,"g of (3-s it oste rol/ml. Cell s were grown mon­ For C RC-IV-'I detection in Western blots, cell s were injectcd in boiling 10% oxc ni ca lly by inoculation with EJ/(erobllcler aerogenes or in a steril e syn th eti c SOS containing protease inhibitors. These contained '15 I"M pepstatin A and 42 medium (4 1). ~M Pefabloc (Serva, Heidelberg, Germany), 100 ~M leupeptin. and 28 ~M E64 Comllutational analysis. BLAST searchcs werc performed according to thc (Biomol, Hamburg, Germany), 75 mU of aprotinin/ml , 'JO ~M chymostatin, and mcth od of Altschul et al. (3) by usi ng either the NCB! database (http://blast. ncbi 10 ,"M antipain (Sigma, Munich, Germany). After completing witb tc nfo ld­ .nlm .nih.gov), the Paramecillm genome databHse (http://paramecillm.cgm.cnrs-gif conce nt ratcd sample buffer, samples we re direclly su bjected to SOS-PAGE. .fr) or thc transporter protein analys is database TransportDB (http://www Immunolocalizalion and affinity labeling studies. Immunolabeling wi th Abs .membranetranspon.org). For dHtabascs used in addition for other genomes sec against CRC-IV- I (R772 and R883), POI, and V-type H+-ATPase was per­ Table S4 in the supplement al material. Protein alignm ents we re performed with formed as descri bed prcviously (53). Labeling of trichocyst matrix proteins and CLUSTAL W (94). Phylogenetic and moleculHr evolutionary analyses were carried a-tubulin was performcd by permeabili zation of knockdown cell s for 2 min with out by usi ng MEGA version 3.0 (50). Predictions of mcmbrane topologics wcre 1% Trit on X- IOO in PI-lEM buffer 160 mM PIPES, 25 mM N-(2-hyd roxyethyl)­ performed with HMMTOP 2.0 (97) or with the TopPred II algorithm (19). piperazinc-N'-2-ethanesulfonic acid (HEPES), '10 mM EGTA (ethylenc glycol Determination ofCRC-IV-1 sequences. To determine macronuclear sequences tetraaceti c acid), 2 mM MgCI2 (pTI 6.9)). foll owed by a fl xa ti on period of 10 min of CRC-IV- Ia, we screened a gc nomic li brary of P. lelrallrelia according to thc in 2% formaldehyde dissolved in PHEM buffer. Formald ehyde WHS rcmoved by method of Ke ll er and Cohen (42). Full-le ngt h sequ cnces of C RC-IV- Ia and two washing steps with modified T ris-hulfered saline (111 TBS) containi ng 10 111 M CRC-IV- 'II> were amplifl ed with the AccuTaq LA D NA polymerase (S igma, T ris-I-ICI, 15 mM NaCl, 0.1% Tween 20, and 3% bovine serum albumin (pH 7.4). Munich, Gcrmany) using 50 ng of macronucl ear DNA as a template (for prim ers, T hen cell s were in cubated for 30 min wi th primary Abs di luted in mTBS and 3607

after two washing steps with secondary Abs diluted in mTBS. We applied Alexa tropiloresis. PCR ampli fi cation data we re coll eetcd wi th the Bio-Rad iCyeler iQ Fluor 488 or 594 F(ab'), fra gments of goat anti-rabbit IgG, or Alexa Flu or 594 real-tim e detection system so ftware and analyzed using the threshold cycle (e,) F(ah'lo fragments of goat anti-mouse IgG (H + L; Molecular Probes, Euge ne, relati ve q uan tificatio n meth od as desc ribed by Li vak and Schmittgen (55). For OR) diluted I: 150 in phosphate-buffered saline- I % bovine serum albumin. Fi­ normalizing th e data we used actin 1-1 (aetl -'I; accession no. AJ537442) as the na ll y, cell s were washed twi ce with mTBS and mounted with Mowiol for confocal reference gene, and data were calculated using th e relative expression with mi croscopy. Membrane staining wit h 3,3'-d ih exaoxaearbocyanine iodide respect to aetl-I as 100%. (DiOC,,; Sigma) was performed after Ii xat ion at a Iinal concentration of 0.1 ",g Physiological tests, To assay the sensitivity of silenced cells to diffcrent of DiOC" per ml of phosphate-buffered saline for I h. [Ca" I... cells were in cubated in feeding solutions supplemented with I mM Conventional fluorescence microscopy was carried out on an Ax iovert 1001V EGTA to yie ld different [Ca2 +lu valu es adjusted by adding 1.1 , 1, or 0.85 mM

(Ca rl Zeiss, Jena, Germany) equipped wit ll Ouorescein isothiocyanate (F!TC)­ CaCl2 • Free ICa ' I 10 was caleulated according to the method of Patton et a l. (70) Filterset 9 aod with a ProgRes ClO plus camera (Jenoptik, Jena, Germany) . using th e MaxChelator program Winmaxe v.2.40. Cell s were counted daily, and Images were captured by usi ng ProCa 2.0 software (Carl Zeiss). Confocal mi ­ division rates we re determined by calculating the number of cell fissio ns per day. croscopy was carried o ut with a LSM 5 10 META using a x 63 Pl an-A pochromat Capability of trichocyst exocytasis was mimicked hy adding an excess of saturated (NA = 1.4) objective (both Carl Zeiss) . Images were processed by using LSM picri c acid (79). The pumping cycles of contractile vacuoles were determined by image browser (Carl Zeiss) and further edited with Adobe Photoshop (Adobe measuring the tim e between contracti o ns of the central vacuoles of cells con­ Systems, San Jose, CAl. tain ed in a mi crocl rop ove rlaid with paraffi n oil. 1lIIlIIlIno-clcctl"0I1 lIIinoscopy 10calizatiol1 , T he method applied is as speci li cd Ca2+ Huorochrome analyses, Ce ll s were immobili zed and loaded by mi eroin­ by Kissmehl et al. (45). I n brief, cell s were injected into 8% formaldehyde plus jection with the Ca" llullruchrulI1 c, Fum R ed, as previllusly descri bed (47). 0.1 % glutaraldehyde (pIT 7.2) at O°C, with a quenched-now device, and processed Intracellular concentration of Fura Red was - 50 ",M. For excitation, two wave­

"" " by the "progressive lowering of the temperature" mcthod. We reduccd the lengths were used (1. 0 ,0;, = 440/490 nm), and the ratio of emission (1. 0 = 650 temperature stepwise, with in creasing ethanol concentrations. This was foll owed nm) at bo th wavelengths was calcul ated. This allowed the analysis of Ca" by LR Gold embedding and UV polyme ri zati o n at -35°C, CRC-IV-I specific in depc ndently of nuorochroll1 e concentra ti on in the cell and of ce ll shapc Abs (R883) have been used for locali zation by protein A-gold conjugated to changes. T he area evalu ated, 3 by 10 t ~ m , was o rie nted parallel to the cell surface 5-l1m gold (pA-Aug) in a Zeiss electron microscope, E MIO. at the cortical region. The ratio analysis was then transformed into fl[., valu es, Gene silencing constructs. Tandem constructs representing both isoforms, whi ch means th at any Ouo reseence readin gs during stimulation (j) were re ferred

either of the IP] R N or of the CRC-IV- I gene families, were cloned inta the to the reading before stimulati o n (f,,). T he f/f" rati os were expressed as apparent double TI-promoter plasmid pL4440 described by Timmo ns and Fire (95), here ICH 2+ Ii increase valu es, starting from I as a representative resting value of designated as pPD vector. An 1,1 70-bp eDNA fr agment of IP]R N- I was ampli ­ ICa2+ I,. Measurements were made at a Zeiss ICM 405 microscope.

fi ed hy PCR and cloned witil Xil ol and Pstl. IPJ Rw 2 speci fi c sequences were All trigger agents were diluted in 5 mM PIPES (pI-1 7.0). The actual concen­ added by introducing a I,O'19-bp pCR fragment using Mlul and Pstl. For CRC­ tration of th e trigger at th e ce ll surface was estimated from the dilution as IV-J recepto rs,' a S72-bp PCR-amplified eDNA fragment of CR C-IV-Ja and a pn:viuusly described (47). T he final ex tracellul ar Cllnccntrations or AED (7~) , 945-bp eDNA fragment of CR C- IV- Ib (for primers, sec Tahle Sl in the supple­ caffeine (48), and 4-CmC (46) were 2 ", M, 50 mM, and 0.5 mM, respectively. Low mental ma te ri al) were lirst subcluned in tb e pCR2. 1 1'01'0 vector (Invitrogen). vaill es of [Ca'+ I" were achieved by adding the ultrafast Ca2 + chelator BAPTA Cloning into th e pPD vector was performed with Xhol and Spel for CRC-IV- Ia (final co ncentra ti on, 1 mM) to th e trigge r solut io n to yield a [Ca" I.. of - 30 nM, sequences, fo ll owed by insertion of CRC-IV-Ib sequences using Xhol and Kpnl. i.e., slightly below ICa2+ I, at rest (- 65 nM 147 J) . This a ll owed us to monitor

The plasmidS\"ere designated pPD-N I N2 (IP, R N specifi c) or pPO-C I C2 (CRC­ selectively the Cal . signal resulting from the release from cortical stores and 2 IV-1 specific). As a negati ve cont ro l (pPD-GFP), th e coding sequence of green undisturbed by Ca • influx. lIu urescent protein (GFP) da ived fr om til e pI'XV-GFP vector (35) was cloned Accession nllmbers. The Paramecium DB accessio n number for CRC-IV- Ia is hy using Kpnl. PTETO l3800001001. C RC-IV-Ib seque nces were submitted to the EMBL da­ Gene silencing by feeding. pPD constructs containing open reading frames of tahase and arc avai lable under accession nllmber BNOOl 236. eitile r OFP (pPD-GFP, negative contro l), IId7 (non-discharge 7 gene [881, pos­

itive control) or parts of both isoforms of JP, R N (pPD-NI N2) and of C RC-IV-I (pPD-C IC2), respectively, were transformed in to E.co!i strain HTI1 5 (DE3). RESULTS Single colo ni es were picked and haeteria were grown to an optical density at 600 nm of 0. 25 in lysogeny broth (LB) medium supplemented with tetracycline ( 12 Identification of genes related to mammalian IPJ and ryan­ tJ.g!m l) and ampicillin (100 ",g!ml) and double-stranded R NA sy nthesis was odine receptors. We screened the P. tell'aurelia genome (4, 5) induced in the presence of 0.5 mM IPTG (isopropy l -~-D-thiogalactopyrano ­ for identifying putative genes related to mammalian intracel­ side). Afte r 3 h of induction, bacteria were harvested by centrifugati o n (1 5 2 + min at 4,000 X g) , and the LB medium was completely removed. Feed ing lular Ca release channels of the IP3 or ryanodine receptor solutio ns were prepared by resuspending bacteria in SM medium supple­ type, In detail, we performed a homology-based database mented with ampicillin ( 100 ",g/ml), ~-si t os t e rol (0.4 ",g/ml), and 0.5 mM search with sequences corresponding to channel domains of IPTG. After adjusting the opti cal density at 600 nm to 0.2, feeding solutions known IP3 Rs and RyRs, the RIH (RyR and IP3 R homology) were applied to Paramecilllll cell cultures afte r sta rving for 3 h in PIPES consensus sequence and the IPJ-binding domain of Mus mus­ buffe r (pH 7.0; 5 mM PIPES, I mM KCI, '1 mM CaCI, ). Cell s were daily counted and transferred to fr eshly prepared feedin g solutions culus IP3Rl. After authentication of the received sequences by by adjusting th e cell numbe r to 50 cell s per ml. For single cell analyses, Pam­ BLASTP analyses using the NCB! database and InterProScan mec illlll ce ll s were separated in depression we ll s containing 100 111 of feeding domain analyses (107), the Paramecium genome yields 34 solution. genes coding for possible CRCs (Fig. 1). Phylogenetic analyses Analyses of RNA from silenced cells, Total RNA fr om silenced ce ll s was prepared hy either by usin g the High-Pure RNA iso lation kit (Roche Diagnos­ reveal that the 34 candidates can be divided in six groups (Fig. ti cs) o r, for sin gle cell analyses, the RNeasy micro kit (Oiagen, Hilden, Germany) lA), each showing characteristic features regarding their rela­ according to the manufacturers' protocols. cDNAs were obtained by using the tionship to IP3Rs or RyRs. As determined by BLAST analyses OuantiTect reverse transcription kit (Oiagen). (3), the positions of conserved regions of individual CRCs (Fig, Ampl ifi cati on and quantificati on of cDNAs were performed by real-time PCR IB; for details, see Table S3 in the supplemental material) in the Bio- Rad iCycler iO real-time pCR detecti on system. Reactions were carried o ut in a fina l vo lum e of 25 ",I using 2 ",I of cDNA, 2 X iO SYBR Green mostly overl ap within one group, th ereby confirm in g th e di s­ Supermix (Bio-Rad,; Munich, Germany), and 12.5 pmol each of forward and position of the 34 genes into six groups, reverse primers (sec Table S I in the supplemental material). Thermal cycling was At a first glance, regardin g the sizc of th e CRCs identified in initia ted with a first denat ura ti on step of 3 min a t 9SoC, foll owed by 45 cycles of P. tetraurelia , all of them seem to be more related to IP ~ Rs than 95° for 20 s, 54°C for 20 s, and 70"C for .10 s; tbe ampl ification Ou o reseenee was read at 70' C at the end of the cycle. All primer pairs were tested with maero­ to RyRs, since none of them reach molecular weights known nuclear DNA and eDNA, and in a ll cases a single amplicon of th e appropriate from metazoan RyRs consisting of roughly 5,000 amino acids 1l1l.: lling temperalure and size was ve rified by ui ss ucia ti oll curves and gel dci,;- (29, 104). However, simil arities to RyRs are restricted to C- 3608

A CRC-I-1a (Se9A) CRC-I-1b (Se26) Se9A (2519aa) CRC-I-1e (Se98) Se26 (2977aa) 1Group I: CRC-I-2a (SeiO) se98l3036aa) IP3R I RyR- '-----:-:10:-::10 CRC-I-2b (Se32) 2999aa) related CRC-II-1 bl se34) IP3RN2 3009aa) 100 CRC-II-1a Se48) IP3RN1 ,------CRC-II-2 ( e30) I:IIjF'€~=.:i:I:!§9i:====2i'i1,,~. 'iiii""'iE""iir;...a. MmlP3R1 (2749aa) CRC-II-31se144) IP3RN2 (2888aa) ,--- - - CRC-II-4 Se1) Group II: L-___ CRC-II- (Se79) IP3RN1 (2890aa) Se30 (3017aa) IP3R·related RIH-domain ,----__---" 1""'1 CRC-III-1a (Se86) Se144 (2886aa) CRC-III-1 b (Se158) ] IP3-binding Se1 (2910aa) domain CRC-III-2 (Sel5) Se79 (2847aa) ,-----CRC-III-3 (Sel8) CRC-III-4e (Se37) Se86 (2595aa) CRC-III-4b ($e17) Se158 (2583aa) Group III: CRC-III-4a (Se4~) Se35 (2059aa) IP R-related Se38 (3036aa) 3 IP3-blnding r--______-'.! 10"'l0 CRC-IV-1 a Ise138) Se37 (2832aa) domain CRC-IV-1b Se80) Se17 (2822aa) ,-----CRC-IV-2 (Se98) Se49 (2835aa) '------:cd CRC-IV-3a (Se62) CRC-IV-3b (Se24) 30aa) CRC-IV-4a (Se6) CRC-IV-4b (Se3A) MmlP3R1 '------~5OL---.:== DmIP~R 50 100 CelTR1 CRC-V-1 (Se2) CRC-V-2 (Se3C) ,------CRC -V- ~ (Se38) '------:-::-::1 CRC-V-4a (Se96) CRC-V-4b (Se106) ,------CRC-VI-3 (Se134) 76L ___ __d-----:= CRC-VI-1 (Se448) Group VI : 100 CRC-VI-2b (Se44A) IP3R-related CRC-VI-2a (Se18) ] ?

FIG. !. Ide ntification of 34 Paramecium CRCs related to IP, Rs and RyRs. (A) Evolutionary relationship of putative CRC proteins from Paramecium. Predictions from multiple sequence alignments are shown in a neighbor-joining tree with 1,000 bootstrap replicates generated with the MEGA version 3.0 program (50). Sequences representing three different metazoan IP3 receptors are from Mus musculus (ide ntified as MmIP, R] in th e li gure; accessiun nu. NP_034715.2), Drosophila melanogasler (DmIP)R, accession no. BAAI4399.1), or Caenorhabditis eleglll1s (CeITR1 , accession no. NP _001023173). Accession numbers of th e Paramecium sequences are summarized in Tabl e S2 in the supplemental materi al. Bootstrap support values are given at th e branches, and evolutionary distances are indicated by the scale bar below. "Sc" de notes scaffo ld numbers according to macronuclear DNA sections as designated in the P. letraureiia genome database (http://paramecium.cgm.cnrs·gif.fr). (B) Sequence analysis of putative CRCs from P. lelraurelia and their relati onship to RyRs (represented by CeRyR, accession no. BAA08309. 1) and IP, Rs (represented by MmIP, R1, accession no. above). Gray bars represent regions homologous to IP, Rs; regions homologous to RyRs are shaded in green. Positi ons of fl anking residues of putative channel domains are highlighted in red and TPr binding domains are highlighted in blue. Conserved regions were determined by BLAST analyses (3) (dark gray bars) and extended by using th e BLAST 2 SEQUENCES tool (92) (light gray bars and green bars). Pusiti ons o r il anking residues or conserved CRC sequences, as we ll as curresponding e-valu cs arc outlined in Table S3 in the suppleme ntal materi al. RIH, RyR and IP)R homology domain according to Ponting (80); Sc, scaffold number.

terminal parts of the proteins, and only N-terminal parts of mologous to IP3 Rs. One gene (P. LeLraurelia lP3 R ;v" l), repre­ group I members share consid erable similarity to RyRs (Fig. sented by M24E11u(rc), was recently characterized in detail

IB). The relationship to IP3Rs is most pronounced in group I (53) (accession no. CR932323), whil e the present work envi s­ and II proteins, wh ere a central RIH domain could be identi­ ages unraveling the gene represented by M22Holr. First, we fi ed in all group members. CR Cs of group III share homolo­ have screened an indexed genomic library (42) in the lab of gous regions to IP3 Rs covering almost the complete receptor Jean Cohen (CNRS, France), all owing us to identify 11 clones sequences, but none of them possess a RIH domain, wliich covering 8,892 bp of the gene. Residual sequence information again emerges in some members of group V CRCs. In contrast was also obtained from the still ongoing Paramecium genome to group II proteins, the RIH domain of group V members project organi zed by Genoscope (http://www.genoscope.cns appears in N-terminal parts of the proteins. Group IV proteins .fr). The cDNA sequences were cloned and analyzed, resulting show an aberrant architecture, as IP3R- and RyR-related re­ in an ORF with 8,991-bp coding for a protein with 2,997 amino gions seem to be restricted to a highly conserved chann el acids and a calculated molecul ar mass of 351 kDa. Comparison domain so th at th e N-termin al parts of th ese pro teins clo not fil of the genomic sequence with their cDNA equivalent resul ted the kn own pattern of IP3Rs or RyRs (Fig. IB). in two introns each of 25 bp (Fig. 2A). Based on the relati on­ Molecular characterization of CRC-IV-! channels. Previ ­ ship to the other Paramecium CRCs (Fig. 1), we coul d classify ously two partial sequences or CR C genes could be id entified this gene as a member of group IV CRCs belonging to a family in a pilot genome survey of P. leLralirelia (21), implying two of two closely related isoforms. The gene was designated eRe­ clones, M22Holr and M24E11u(rc), containing sequences ho- lV-l a and the related isoform as eRe-lV-lb. Both isoforms 3609

A B PtCRC-IV-1 a ATG(+1) PtCRC-IV-1a T~t(+9044) N ==(2=9=9=7=a=a=)======A~B'!Ii:Ech=.nZinc· lpi C 260& k880 1331 .703 Intra" S' 1316 1727 Intro" 3' RnlP3R3 ATG(+1) PtCRC-IV-1b TGA (+9052) Rn: 1872-2609 At 30-47 8343 8713 Inlron 6' PI: 2240-2662 2744-2966 MmRyR1 Inl 1735 Intron 3' Mm: 3887-4224 481 3-5021 1e·7 9e-25 PtCRC~V - 1" 1-2997 PtCRC-IV-1 b PrCRC- IV-1b: 1-3000 .=0.0 Pt~ -!;.!!.!.... __~ 1325-2181 n: 2-584 708-1085 1310-2947 3e-09 2e-32 20- 143 ~J2.;!!!._..:.;66~6-.:.;:10;:.:33_ 1621-2873 TC00762860 71: 38-359 474-9 11 1064-1256 1404-2874 2e-l1 60-26 3e-07 30-157 P';~ ___8'!!;.l.228 1521-2409 2620-2903 TC00138560 I II III IV V P VI 71: 37·420 805-975 1467·2336 2429-2806 2606-2626 2635-2655 2692-2712 2717·2737 2765-2785 281 0-2830 2860-2880 2&-18 28- 10 38-55 4e-62 o ~P J Rl 2439 DmIP3R 2527 CeITRl 2569 ~yR l 4829 DmRyR 4906 CeRyR 4867 PtIP3RN- l 2632 PtIP3RN- 2 2630 PtCRC-IV- la 2765 KD~~gg------EV PtCRC- IV- lb 2 769 KE ------EA ~P , Rl 2519 DmIP, R 2606 CeITRl 2637 ~yRl 4867 DmRyR 4937 CeRyR 4900 PtIP, RN- l 2661 PtIP, RN- 2 2659 PtCRC- IV- la 2807 PtCRC-IV- lb 2810 FIG. 2. Molecular structure of the Paramecium CRC-IV-l channels. (A) Schematic representation of both isoforms of CRC-IV-i genes from P. te/m ureliil . Start (+ 1) and stop (+ 9044) codons of CRC-IV-iil were determined by RT-PCR, as were th e positi ons of introns (triangl es), while CRC-IV-lb was determined on the basis of homology. (8) Schemati c representation of CRC-IV-Ia. The putative channel domain (L2606 to 0 2880) is shaded in gray; "Ab" denotes the antigeni c region (P2457 to 1-1 2556) used to raise polyclonal Abs. Alignments showing CRC-IV-la regions ho mologous to RnIP3 R3 and M. musculus RyRl are listed below, as well as to P. letrat/relia CR C-lV-tb and th ree orthologues identifi ed in T thermophilil (TTI-I ERM_00IJ8560, TTI-I ERM_00762850, and TTHERM_00762860). Positions of residues referring to CRC-IV-la sequences are highlighted in boldface type above the traces (P. lelrilurelia [P/]) , wh ereas pos iti ons of residues referring the homologous counterparts (Rilttus nOlvegicus [Rn], M. musculus [Mml , and T. Ihermophi/a [Tt]) are in dicat ed with th e corresponding e-valu cs below. (C) Hydropathy pro fil e of the channel domain of CRC-IV-'Ia (F2580 to 1-1 2920) by Kyte-Doolittle scales (52) reveals six highly hydrophobic segments. Putati ve positions of transmembrane and pore regions (I to VI , P) were predicted by using the TopPred II algorithm (19). (D) Mu ltiple sequence ali gnm ent of putative pore regions (highlighted in red) with adjacent transmembrane segments (highli ghted in blue) of Pammecium CRCs IP"Rw l (accession no. CAI39'149), lP, Rw 2 (accession no. CAI39 148), CRC-IV-la, and CRC-IV-lb in comparison to metazoan IP3Rs (Mus musculus IP, Rl, Drosophila melanogaster IPJR, and Caenorhabdilis elegans ITR) and RyRs (Mus musculus RyRl , D. melanogasler RyR [accession no. BAA042121, and C. elega f/ s RyR rac cession no. BAA08309]). Residues that are identical are shaded in black; similar residues are shaded in gray. The amino acids highlighted in ye llow wi th in th e pore segment correspond to putati ve selecti vi ty filt er regions (Jl). Tn RyR sequences the glutamin e critica l for ryanodine interaction is marked in green (100). Predictions of membrane topologies were pelf ormed with HMMTOP 2.0 according to the method of Tusillidy and Simon (97). share 83.3% identity on DNA and 89.7% identity on amino and TMD6 (62). Two parts of the CRC-IV-l C termini could acid level. Sequence comparisons with the Paramecium data­ also be aligned with sections of RyRs. One part in each isoform base are shown in Fig. Sl in the supplemental material. (CRC-IV-l a 12240-V2562 and CRC-IV-1b 12247-F2585) over­ According to their molecular topology, group IV members, laps with a region responsible fo r RyR activation by 4-CmC CRC-IV-l a and CRC-IV-lb, are conse rved regarding their (26, 27) (sequence alignments are shown in Fig. S2 in the C- terminal domains, which show high agreement with corre­ supplemental materi al), the other part (CRC-IY-la D2744- sponding regions of mammalian IP3Rs (Fig. 28). Analysis by P2965 and CRC-IV-1 b D2748-P2968) matches with RyR pore­ Kyte-Dooli ttle scales (5 2) reveal that CRC-IV-1a and CRC­ forming segments. Focusing on the pore region, sequence IV-lb (data not shown) possess six hydrophobic stretches alignments show th at the Paramecium CRC-IV-l channels, as within this region (Fig. 2C). Thus, they resemble IP3 R channel well as the recently described P. tetraurelia IP3 R N -l receptor domains, which consist of six transmembrane spanning seg­ (53), and its closely related isoform P. telraurelia 1P3Rw2 (see ments (TMDl to TMD6) with the pore regi on between TMD5 Fig. 1) are related rather to RyRs than to IP3 Rs. As shown in 3610

Fig. 2D, the Paramecium CRCs, just like me tazoan RyRs, lack A COOM CRC-IV-1(R883) PIS B .-___+--,EDTA the extended luminal loop region between TMD5 and the 2 I ngAG 2500 '50 10 250 pore, as it occurs in metazoan IP3Rs. The putative selectivity kDa kDa filler motif GGGI/vGD within the pore regions of RyRs a nd

IP3Rs (9, 11) is conserved in CRC-IV-1 channels, whereas the 0- glutamine present in C-terminal transmembrane segments of 212 RyRs critical for ryanodine interaction (100) is not present in 170 CRC-IV-1 channels. 116 N-terminal parts of CRC-IV-l proteins are less conserved 76 53 a nd only sequence searches of the genome of th e related ciliate Tet.rahymena lhermophila (23) revealed three genes (TTHERM_00138560 [GenBank accession no. XP _001019 838.2], TTHERM_00762850 [XP_ 001025343.1], TTHERM_ 30 00762860 [XP_001025344.1]) , which share homologolls rc­ 20 -- gions covering the complete CRC-IV-1a protein (Fig. 2B). This suggests that these kinds of CRCs might be ciliate specific. Thus far, we we re not able to identify orth ologues in a ny other ciliate using the Ciliate Ortholog Database (http://oxytricha.princeton.edu/COD/index.html). CRC-IV-l 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 polyclonal Abs (R772 and R883) against a recombinant peptide compris­ ing residues P2457-H2556 of CRC-IV-1a. This region shares 25 85 % id entity (e = 5 x 10- ) to the corresponding region in CRC-IV-1b, suggesting that both members of CRC-IV-1 chan­ nels are recognized by the Abs. Considering any poss ible cross­ reactions of the Abs, sequence analyses were performed. This reveals that the CRC-IV-1a peptide P2457-H2556 shares no significant sim il ari ty with any oth e r CR C proteins. Only some additional group IV members show weak conservation. In de­ tail, identities are as follows: CRC-IV-4b(Sc3A), identities 19/63 (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), ide ntities 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 for CRC-IV-1a and -lb. In Western blot analyses, alfi nity-purifi ed R883 Abs recog­ nize th e CRC-IV-la peptide P2457-I-I2556 with high affin ity (Fig. 3A) and detect a high-molecular-weight band in PG/'ame­ cium whole-cell homogenates, which seems to be stabilized through the presence of divalent cations (Fig. 3B). Immuno­ nuorescence labeling with th e same Ab results in a strong staining of a network throughout the cell (Fig. 3D) and of regul arly arranged plate-shaped structures in the cell cortex (Fig. 3C) clearly representing a lveolar sacs for reasons speci­ fied below and in th e Discuss ion. Colocali zation studies with

(B) Western blot ana lysis of Paramecium whole-cell homogenates us­ ing Ab R883 reveals two bands of - 250 and - 30 kDa (arrows). The discrepancy from th e value expected may be due to partial degrada­ tion. Samples were prepared in the presence (+) or absence (- ) of FIG. 3. CRC-IV-I is loca li zed in the ER and in alveolar sacs. EDTA. (C and D) Confocal images of a ce ll stained with anti -CRC­ (A) Purified Abs against CRC-1V-I (R883) recogni ze th e polypep tid e IV-I Abs (R883; red channel) and with DiOCr. (green chan nel) show corresponding to CRC-IV-la residues P2457 to H2556 with high af­ co loca li zation of both markers in the ER, but not in th e alveolar sacs, finity (lanes 2, 3, and 4) in il11l11l1ll oblol S, whereas th e rreil11l11l1lle which were labe led only with R883 Abs. (C and D) Confoca l sli ces se rum (PIS) yields no signal (lane 5). Lane 1 (COOM), Coomassie were taken from surface (C) and median section s (D) of the cell. oc, blue 5t,'ining or purified polypeptide (2.5 '.lg) used for immunization. oral cavity. Scale bars, 10 J-I.m. 361 1

FIG. 4. Colocali za tion studies using Abs agai nst CRC-TV-1 and the ER reside nt POl. Confocal im ages of a cell stai ned with purified polyclonal Abs against CRC-IV-l (R772; green channel) and POI (red channel). (A) Staining of the alveolar sacs exclusively by R772 Abs is shown by confocal sli ces taken from the cell surface. (8) Details are evident in an enlarged image. (C) In a medi an section plane, colocalization of both Abs occurs only in the ER. oc, oral cavity; cp, cytoproct. Scale bars, 10 flm.

DiOCr" a marker for the ER applicable to Paramecium (82), the ce ll cortex (Fig. 4A and B). According to their position at show that the DiOC6-labeled meshwork overlaps largely with the cell periphery, shape, size, and arrangement, these struc­ the R883 staining in the cell interior (Fig. 3D), suggesting that tures correspond to alveolar sacs. A detail ed ana lysis by im­ CRC-IV-l chann els also reside in the ER. This finding was munogold electron microscopy indicates not only that CRC­ endorsed by costaining with a polyclonal mouse Ab (PDI; for IV-l channels are present in these cortical Ca:!+ stores, but AB characterization, see Fig. S3 in the supplemental material) , furthermore that labeling is restricted to membranes at the which was raised against a recombinant peptide corresponding outer sid e of alveolar sacs facing the cell membrane so that any to Paramecium PDI 1-1 (PDIl-l; accession no. CAD99202) Ca2 release would be spilled over trichocyst exocytotic sites residues L33-E145, and th e CRC-IV-l speeili c Ab R772. As (Fig. 5). shown in Fig. 4, doubl e labeling occurred in the ER network Gene silencing by feeding. Knockdown in Paramecium can (Fig. 4C), while R772 Abs sole ly stain scale-like structures in be achieved by feeding transform ed bacteria which produce

FJG. 5. Tmmunogolcl electron mi croscopi c locali zation of CRC-IV-l. Imillunogolcl iabelin g with CRC-IV-J-specific Abs R883 (arrows) occurs selectively on the outer part of the alveolar sacs (as) facing the cell membrane (cm). t, clocked trichocyst. Bar, 0.1 flm. 3612

tion of the Paramecium actl-l (accessi on no. AJ537442) was A N1 (258-361) C1 (5176-5280) used for data normalizati on and as an inte rnal control for ~ (255--358) ~ (5211 -5327) signals originating from any possible contaminating DNAs dur­

ATG in g the amplificati on ste p. As show n in F ig. oB, mRNA levels of CRC-IV-1 channels are reduced in pPD-C1C2-treated cells " CRC · IV~1a (872bp) ,---CRC.IV-1b (945bp) ," ~~;===~==~~~~~~~~~~pPD-C1C2 without affecting expression ofIP3 R N receptors, whereas treat­ : 7553 8424 7496 8440 ment of cells with the pPD-N1N2 construct leads to decreasing : 1P3RW2 (1019bp) ~ t sssssssl\ts~1U~Z~tssssssss , I pPD .. N1N2 tra nscript levels of IP3 R N receptors. We observed only slightly 9\ 0 1928802 1971 reduced CRC-IV-1 transcript levels in pPD-N 1N2-treated ====:::::l1+728 bp ORF+I=I ======:::1' pPD-GFP cell s, whil e we can co nArm th at th e feedin g constru cts pPD­ NIN2 and pPD-CI C2 each lead to a specifi c knockdown of ====:::::ll".. f---- 1518 bp ORF---"~ ~ pPD-ND7 their respective target genes. To confirm th at diffe re ntial RNA ex pression is para ll eled by B 120 ,------. concomitant protein concentrations in silenced ce lls, we per­ fo rm ed im mun oflu orescence analyses. For compa ri so n, slain­ ing procedures and im aging processing of silenced cells and mock-treated cells were performed under identica l conditions. As shown in Fig. 7 A and B, a strong reduction of R 772-labeling of almost all CRC-IV-1-s ilenced cells (pPD-ClC2) could be observed compared to control cells (pPD-GFP), whereas la­ beling with Abs against cx- tubulin still persists at the same level. Notably, the reduced R772 staining is not correlated with o C1 C2 N1 N2 structural alterati ons or modifi ed arrangements of alveolar sacs in CRC-IV-l-sil enced cells. Similar downregulation could • pPD-GFP o pPD-C1C2 o pPD-N1N2 be obtained, with the respective Abs, for IP3 R N receptors in FIG. 6. Sil encing of CRC- IV-l and IP3RN genes by feeding. gene-silencing experiments. Since these CRCs localize to the (A) Scheme of the silencing constructs for both isoforms of CRC-IV-l CVC (53), cells silenced in these receptors throug h treatment and IP3 RN gene famili es represented by pPD-CIC2 and pPD-NIN2, respectively. Length and positions of the cl oned fragm ents refer to with the pPD-N1N2 construct display a reduced CVC labeling cDNA sequences of eac h CRe. As a negative co ntrol, we app lied the compared to control cells (pPD-GFP) when stained with ORF of GFP cloned in the pPD vector (pPD-GFP). As a pos itive IP)RN-specifi c Abs (R866; Fig. 7C), while the CVC still could co ntrol, the pPD-ND7 construct was used, whi ch contains the ORF of be stained with Abs against the V-type H +-ATPase (Fig. 7D). the ml7 gene impairing tri chocyst exocytosis. Cl and C2 or Nl and N2, As a marginal result, we only observed extreme ly swoll en am­ highlighted in red, indicate the positions of CRC-IV-l a (Cl), CRC­ pullae or, in some cases, more severe perturbation of the IV-lb (C2), IP3Rw1 (Nl) and IP3RN -2 (N2) specifi c amplicons used fo r real -time RT- PCR. (B) Expression pattern of CRC-IV-l and organelle (Fig. 7D). IP3RN reccptors during gene sile ncing. Total RNA (0 .5 I-lg) was iso­ General effects of gene silencing on cell activity. In a fi rst lated from - 4,000 Paramecium ce lls mock silenced with pPD-G FP, approach, we examined whether silencing of IPJR or CRC­ pPD-Cl C2, and pPD-N1N2, each for 72 h. The relative abundances of N IV-I receptor families has an effect on Paramecium cell divi­ mRNAs of CRC-IV-la (Cl), CRC- IV-lb (C2), IP,R -l (Nl), and N sion. Tn gene ral, a sli ghtly reduced divisio n rate during th e first IP)RW 2 (N2) were determined by rea l-time RT-PCR. The relative express ion was ca lcul ated by using the t:J.t:J.C.,. method, and expression 24 h of sil encing could be observed, probably due to a lag effect levels are give n as the percentage of tra nscripts norm ali zed to the after transfer from normal medium to feeding solution (86). actl -l messe nge r. Th e data are means ± the standard error of the 2 F urthermore, Ca + concentration in the culture medium af­ mean (S EM) from three repli cate wells. fects division rates of sil enced cells. When [Ca2 +]., of the culture medium was adjusted to 100 fLM , which equ als stan­ dard conditions (76), cell div ision was not influenced by th e double-stranded RNA ho mologous to secti o ns of the gene of sil encin g constructs (Fig. 8A). However, when [Ca2+ L was interest (30). To achi eve a knockdown pheno type of whole decreased to 1 fLM , a significant reducti on of di vis ion ratcs of receptor fa milies, tandem constructs representing both iso­ pPD-CIC2- or pPD-N1N2- treated cells occurred, while con­ forms of IP3 R N and CRC-IV-l gene fa milies were cloned into trol cells treated with pPD-GFP or pPD-ND7 constructs divide the pPD gene silencing vector (see Ma teri als and Methods). just as under standard conditions. A further reductio n of The constructs we re designated pPD-N l N2 and pPD-Cl C2, [Ca2 +L to - 0.3 fL M by e levating the EGTA concentration to respectively (Fig. 6A). As a negative control, the ORF of GFP 1.5 mM prolongs divisio n cycl es even in control cells and, over derived from the pPXV-GFP vector (35, 35) was cloned (pPD­ longer times, causes cell death in pPD-C1C2- or pPD-NlN2- GFP), whereas the pPD-ND7 construct was used as a positive treated cell s within 24 h. Taken together, these expe rime nts control (88), which conta ins the ORF of the "non-discharge" reveal that IP3R N channels and, to a lesser degree, CR C-IV-l gene nd7, resulting in impaired exocytosis of trichocysts. channels are essential fo r viability in media with low [Ca2 +L In order to show that the feeding constructs pPD-NlN2 and for reasons to be a nalyzed in more detail. pPD- I C2 lead to a specifi c knockdown, sil e ncing of th e chan­ We aimed at a more clear distinction between the two CRC nels was monitored by analyzing mRNA levels via real-time types by the following experiments. Considering the locali za­

PCR. Transcript abunda nces were quantifi ed by usin g th e C, tion of IP3 R N receptors to the CVC, we examined in which relati vc quantifi cati on me th od (55). An intron-harborin g sec- knockdown situati on this organell e would be affected. As 36 13

FIG. 7. Immuno labeling of pPO-CIC2- and pPO-N1N2-treatcd cells. Confocal images of a control cell (pPO-GFP) (A) and a CRC-IV-1- sil enced cell (pPO-C1 C2) (B) using Abs against CRC-IV-1 (R772; green channel) and mouse mo noclonal Abs agai nst a-tubulin (OM'IA; red channel). CRC-IV-l labeling of pPO-C1C2-treated cell s is reduced aft er 72 h compared to control cells, whereas th e labeling of basal bodies with Abs against c<- tubulin (see enlargements) is not affected. The general arrangement of alveolar sacs and basal bodies is no t altered in CRC-IV­ I-s il enced cells compared to co ntrol cells. Note th e ideiltical recording conditions for controls and 72-h sile ncin g. (C) J1l11l1un otluorescence analys is of IP3RNl /2-silenced cells (pPO-N1 N2) shows a reduction in labeling of the CVC with IP3 Rwspecific Abs (R866) compared to control ce ll s (pPO-GFP). (0) Stain ing of pPO-NIN2-sile nced cells wi th Abs against V-type H+ -ATPase (V-ATPase [subunit aI-I]) persists, while some sli ght structural alterati ons occur (of which several examples are presented). Most of pPO-Nl N2-treated cell s show expansions of the ampullae (arrows) and in some cells (upper middle im age) the structure of the CVC appears to be less distinct th an normally. Scale bars, 10 1.1. 111 .

shown in Fig. RB, thc fi lling and CXflc lling cyclc is clcarly flro­ (Fig. 8C). In contrast, pPD-CIC2-treated cell s do not show longed from a va lu e of - 10 s in control cell s to > 16 s in such alterations. The contractile vacuole shows regul ar, appar­ IP3Rwsilenced cells. In parallel, in some of these cells the ently even slightly shortened contracti on periods (Fig. 8B), and CVC is severely perturbed as manifested by swollen ampullae no morphological anomaly could be detected (Fig. 80). 3614

A <11JM [Ca2+] ~--~--~~------. 4 0

3 ~ ~ 2 c: o '(jj '::; 24h i5 0 o :o r -1 -1 -- III

L-______~ -2 L::::::===:::::::~ ______~ B 20

2 15 (J

>-(J OJ 10 c: 'a' E 5 :::::I C.

FIG. 8. Additional aspects of gene sil encing. (A) Influence of rCa2+L on IP}Rw and CRC-IV-I-silenced cells. Division rates of sil enced cells were determined by calculating the number of cell fissions per day. Black and dark gray bars represent the division rates of control cells (pPD-GFP and pPD-ND7) and light gray and white bars represent the division rates of CRC-IV-l-silenced (pPD-CIC2) and IP, R w silenced cells (pPD­ 2 NIN2), respectively. The viability of cells treated either with pPD-CIC2 o r pPD-NIN2 is reduced only when exposed to low [Ca +1n compared to 2 2 H 2 control cells at the same rCa +1,.,: n (100 fJoM [CaH ]o) = 40, n ("I fJoM [Ca +].,) = 40, and n «1 fJoM [Ca ]" = 27. P values ([Ca +lu < 1 fJoM): Cl C2 = 4.6181 E - 09 and N1 N2 = 9.84242E- 17. The P values correspond to the Student I test. Error bars indicate the SEM. (B) Pumping cycles of the contractile vacuoles of IP3R N knockdown cells are prolonged (right bar) compared to control cells (pPD-GFP; left bar; P = 0.0003). Pumping cydl:!s 01" CRC-VI-l siil:!I1l;l:! d cdls art: sli ghtly shortl:!nl:! d (pPD-CIC2; middle bar) wi th weak significanct:: (P = 0.01 for pPD-CI C2- versus pPD-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 II (pPD-N1N2) = 43. Error bars indicate thc SEM. (C) Reprcscntative of a live IP3R w silcnced ccll showing extremely expanded ampullae (white arrows) attached to the contractil e vacuole. See also the e nlargeme nt in panel C' . A further conspicuous detail is the swo ll ~n cell body combined with accumul ations of large clystalline inclusions in the cytosol (black arrows). (D) Representative of a live CRC-IV-1-si lenced cell. In contrast to IP1 Rw si lenced cells, the contractil e vacuole is not affected. See a lso the enl argement in panel D'. Scale bars, 10 fJom.

Knockdown of IP]RN and CRC-IV-! receptors all'ect exocy­ also clearly in reduced amounts (Fig. 9A), Under these condi­ tosis of trichocysts, Stimulation of trichocyst exocytosis leads tions we showed th e ability of trichocyst contents to decon­ to rapid expansion of the secretory organell e contents in in­ dense, when Ca2 + gets access (data not shown). soluble needles. The exocytotic capacity of Paramecium cells To visualize more clearly the effects of gene silencing on can be assayed by appli cation of picric acid (79), which acts as exocytotic capacity, we used Abs to stain TMP4, a co mponent a rixative a ll ow in g easy quantirication oJ the amount of ex­ of the inner trichocyst "core" region (98). Immunofl uores­ truded trichocysts (Fig. 9A). cence shows that the amount and the arrangement of tricho­ After 24 to 48 h of treatment with feeding bacteria, both cysts in pPD-ClC2-treated cells is comparable to that observed

IP3R N - and CRC-IV- I-s il enced ce ll s show a sign ifica ntly re­ in control cells (pPD-GFP and pPD-ND7), where trichocysts duced number of dischargeable trichocysts compared to con­ are docked at the cell surface in high density (Fig. lOA). In trol cell s. In contrast to pPD-ND7-treated cells, which display contrast, IP3 R N -sil enced cells display a reduced number of an alm ost complete inhibition of exocytosis, sil encing of the trichocysts, which are mostly not attached at the surface. Sim­ two CRC types analyzed leads only to a partial inhibition, il ar results could be obtained with Abs against TMPl, which individual cells showing different degrees of exocytosis reduc­ recognize the second mature polypeptide of TMPlb and tion (Fig. 9). Partial inhibition in pPD-CIC2-treated cells is thereby decorate the trichocyst "cortex" (98) (see Fig. S4A in manifested by restricted extru sions of trichocysts either in clus­ the supplemental material). This indicates that the general ters along the entire cell body or restricted to the cell poles. In shape of trichocysts is not altered in pPD-NlN2-treated cells. contrast, in IP3 R N -si lenced cells upon stimulation a restricted To co nfirm the results of the iml11un ofl uorescence studies, number of trichocysts is extruded over the whole-cell body, anti-TMPl (see Fig. S4B in the supplemental material) and 3615

gated a possible correlation of trichocyst biogenesis and 2 2 A [Ca "'];, by incubating cell s in medi a with decreas ing [Ca +J.." to which in tracellular concentrations, although at a much lower level, rapidly adjust in these cells (1 2, 24, 43). Via bility at low [Ca2 +]o is demonstrated in Fig. llA, showing also that division cycles are accelerated with rising [Ca2 +J..,. By stepwise lowering [Ca2 +]o levels, the number of di scharged trichocysts decreases gradually (Fig. lIB). To investigate whether the reduced exo­ cytotic capacity results from a reduced number of trichocysts, we prepared whole-cell homogenates and performed Western blot analyses with Abs against TMP4. As shown in Fig. 10C, the amount of TMP4 decreases when cells are grown in media where [Ca2 +lo was reduced to a threshold of tolerable low [Ca2+J..,. This suggests that the reduced number of trichocysts

in IP3 Rwsilenced cells might be a secondary effect of lowered [Ca2 + ]; due to impaired Ca2 + re flu x as a conseque nce of low­ B 10Or------, ered IP3 RN -type receptor levels. Silencing of CRC-IV-l channels results in impaired Ca2+ ~80 2 .!!2 release from cortical stores. Are Ca + transients during stim­ "N 60 pPD-C1C2 ul ated exocytosis reduced in CRC-IV-1-silenced cells? Fura Red-loaded cells were triggered with AED and changes in '0 H :v 40 [Ca ]; were monitored by double-wavelength recordings. As .0 2 2 E the source of Ca + during stimulation includes not only Ca + ~ 20 mobilization from the alveolar sacs but also a superimposed Ca2 + inf"lux from th e medium (46, 72), we pe rformed th e O~~~~~HU~dL~.a~~~ experiments at a [Ca2+]o slightly below [CaH ]; in unstimulated 100 80 60 40 20 o 2 exocytosis (%) cells (see Materials and Methods) to visualize Ca + transients FIG. 9. Knockdown of both CRC types results in reduced tricho­ solely emerging from alveolar sacs (Fig. 12A). Clearly, CRC­ 2 cyst exocytosis. (A) Bright-field im age;:s show representatives of si­ TV-'I sil encin g results in a signifi can t reducti on of Ca + releas­ lenced cells treated with picric acid to mimic exocytosis (see Materials able from stores compared to controls (pPD-GFP). Since and Methods). Trichocysts elonga te upon expulsion and become visi­ former studies had shown that the ryanodine receptor activa­ ble as needl es (several times longer than cilia) attached to the cells. 2 tors, 4-CmC and caffeine, activate Ca + release from alveolar Both cells sil enced in CRC-IV-l (pPO-C1C2) and in IP3 RN (pPO­ Nl N2), respectively, show reduced exocytosis of trichocysts compared sacs (46, 48), we also applied these drugs to mock-treated and to control cells (pPO-GFP). Sil encing of th e N07 gene (pPO-N07), to CRC-IV-1-silenced cells. As shown in Fig. 12A, stimulation 2 our positive control, leads to almost complete inhibition of trichocyst of ce lls with either 4-CmC or caffeine results in similar Ca -!­ extrusion. Scale bar, 10 !-tm. (B) Quantitation of exocytotic capacity transients as observed with AED. Under these conditions, tri­ after 48 h of feeding tested with picric acid. The graph shows quanti­ chocyst exocytosis is almost totally inhibited. Since this, how­ tative distributions of silenced cells with different degrees of exocytosis inhibition. The data were collected in seven experiments. Error bars ever, could theoretically also be due to the requirement of 2 indicate th e SEM. n (pPO-GFP) = 262; n (pPO-N07) = 266; /'I extracellular Ca + for the decondensation and expU lsion of (pPO-Cl C2) = 266; n (pPO-Nl N2) = 241. trichocyst contents (10), we repeated the experiments with 21 CRC-IV-1 sil enced cells in the presence of standard [Ca ' ]., (Fig. l2B). We fo und that upon stimulation with the three anti-TMP4 Abs (Fig. lOB and C) were also used in Western compounds the [Ca2 +]; signals generated are diminished (Fig. blots. As shown in Fig. lOB, the intensity of the band repre­ 12B), and the number of discharged trichocysts in that cases is senting the mature TMP4 polypeptide of ~ 18 kDa is clearly also greatly reduced (to ~ 10%) in comparison to va lues ob­ reduced in preparations from IP3R N silenced cell s compared tained with control cell s (data not shown). This clearly indi­ to extracts from pPD-GFP-, pPD-ND7-, and pPD-ClC2- cates that CRC-IV-l sil encing bears on the Ca2 + signal re­ treated cell s. This indicates that the reduction of exocytotic quired for exocytotic membrane fusion. capacity of IP3 R N -silenced cells is due to decreasing amounts of trichocysts in these ce lls, suggesting that the biogenesis of DISCUSSION trichocysts is affected due to altered Ca2+ homeostasis. CRC­ IV-I -sil enced cells also displ ayed reduced exocytosis but, in Although a plethora of plasmalemmal cation channels are contrast to IP3 RN -s ilenced cell s, the amount and the arrange­ largely characterized in Paramecium (5 '1, 58), identifi ca ti on of ment of trichocysts is not affected. This supports an involve­ intracellular CRCs has remained elusive. Since the Parame­ ment of CRC-IV-l channels in signal transduction. cium genome database has become developed (5 , 21), we Influence of [Ca2 +] .. on trichocyst biogenesis. We hypothe­ started identifying CRCs. As outl ined in Results, these may sized that the reduced trichocyst content in IP3 R N -s ilenced contain some unex pected combinations of domain sequences, 2 cells is a secondary effect due to altered [Ca +];, as IP3 R N recalling in part IP, or ryanodine receptors, while large parts 2 receptors are supposed to be involved in graded reflux of Ca '" are unrelated to any known channel structures. Thus far, we 2 to fine-tune [Ca +]; homeostasis (53). Therefore, we investi- have compared the size, the presence of IP3 binding and the 3616

B r"f,;

37 F======'" 25 20 _ __ TM P4

15 c 120

~100 ~ . ~ 80 $ .S 60

40

20

o TMP4 et-tubulin .pPO-GFP D pPO-C1C2 . pPO-N07 D pPO-N1N2

FIG. 10. Knockdown of IPJRN , but not CRC-IV-l , leads to decreasing amounts of triehocysts. (A) Confocal im ages of si lenced cells stained with Abs against TMP4. Representatives of control cells (pPO-GFP and pPO-N07) or CRC-IV-l-silenced cells (pPO-CIC2) show trichocysts docked in high density at the cell surface. In pPO-NIN2-treated cell s, the overall number of trichocysts and their docking at the cell surface is reduced. Stacks were fu sed from 20 (pPO-N07, pPO-CIC2, and pPO-N"I N2) or 13 (pPO-GFP) confocal sli ces (0.75-fLm thickness). E nl argements show median secti ons to visualize the arrangement of trichocysts at th e cell periphery. Scale bars, 10 fLm . (6) Western blot of protein preparations from silenced cells treated for 72 h with either pPO-GFP, pPO-N07, pPO-CI C2, or pPO-NIN2. As a loading control, the same blot was probed with Abs against et-tubulin (a-tub, upper panel) showing similar protein amounts in each lane. In extracts prepared from pPO-NIN2-treated cells, Abs against TMP4 (lower panc l) rccognize a significantly reduced amoun l of TMP4 in pPO-NIN2-treated ce ll s. (C) O uantitatio n of gray va lues of TMP4 signals in Western blots substantiates reduced TMP4 contents in pPO-Nl N2-treated cells compared to control cells. There is only a sli ght in crease u f' TMP4 signal in pPO-ND7, and a sli ght decrease (alsu nut signifi cant ) in pPD-CIC2-tn:ated ce lls in cumpari sun tu contro ls.

RIH domain structure, putative transmembrane regions, and lar activities. Indeed, our experim ents with control cells at low 2 the nature of the pore domain, as derived from gene structure, [Ca -' ] " also result in a reduced number of trichocysts. 2 but functional analyses have been restricted to CRC-II-1 Theoretically a primary effect of interfering with Ca + ho­ (IP3Rs) (53) and CRC-IV-1 channels (the present study). meostasis by CRC-II-l silencing could be the requirement of a Thus, the channels not addressed in the present study are to be Ca2+ -activated calreticulin complex fo r correct processing and considered only bona fid e Ca2 -1- channels whose properties still fo lding of secretory proteins (20, 38). Although a calreticulin­ have to be established. Subfamily CRC-IV-1 is a novel channel li ke protein occurs in Paramecium (73), we see in Western type. Sin ce both CRC types are invo lved in regul ating distinct blots, after CRC-II-1 si lencing, a correctly cleaved TMP4 pro­ steps of th e secretory cycle in Paramecium, we now compare tein comparable to the size of processed TMPs in normal cells th ese two groups of CRCs. (31, 99). T he small number of trichocysts, mostly remaining Effects of CRC-II-l silencing. These experiments aimed at unattached inside the cytoplasm, observed after CRC-II-l si­ es tab li shing specifi c effects of the (wo P. lelraurelia CRCs un­ lencing may largely represent residual organelles persisting der consideration here. Silencing of CRC-II-l channels causes from before gene silencing. This may be explained as follows. reduced numbers of trichocysts that mostly stay off the cell A sali ent aspect concerning reduced trichocyst numbers af­ membrane, while after sil encing of CRC-IV-1 a full set of ter CRC-II-1 sil encing may concern budding of precursor ves­ trichocysts remains attached to the cell membrane in the ab­ icles and thei r transport and fusion, as this also occurs during se nce of exocytotic capacity. One may take into account dif­ trichocyst biogenesis (99). In higher eukaryotic cells, some of 2 ferent Ca -' -based effects, particu larly since CRC-II-l chan­ these processes, or steps thereof, are known to be subj ect to nels are known to deal with the regulation of basal [Ca2 -1 ]; Ca2+ regulation (1, 6). In P. letraurelia, all steps of exo- and homeostasis (53) . Lack of CaH reflu x from the CVC, whi ch endocytosis are known to be accelerated by in creased [Ca2 -1- ] they were shown to serve, could compromise several subcellu- (72), whereas no information is avail able on trichocyst biogen- 3617

A 3 rc===:::::;------,

-0

-1L-______~

FIG. 11. Biogenesis of trichocysts depends on [ Ca ~+ ] n" (A) Division rates of cells grown in media supplemented with EGTA and CaCI2 were determined by calcul ating th e number of cell fissio ns per day. After 48 h of Ca'+/EGTA treatment, division rates of cells decrease with lowering 2 [Ca +]" (P = 0.036 for 1 mM EGTA-O.8 mM CaCI, versus 1 mM EGTA- 1 mM CaCI,; P = 0.017 for 1 mM EGTA-O.6 mM CaClz versus 1 mM E GTA- 1 mM CaCl,; P < 0.0001 for 1 mM EGTA-O.4 mM CaCl, versus 1 mM EGTA- 1 mM CaCl2 ; P values according to the Student I test). 2 Error bars indicate th e SEM. n. (aliquots of counted cells) = 8. (B) Exocytotic capacity of cells grown for 48 h in media with different [Ca I J.. was 2 quantifi ed by using th e ricric acid test. Note th e in creasing exocytosis with in creasing [Ca +J... II (1 mM EGTA- 1 mM CaCI, ) = 52; n ("I mM EGTA- 0.8 mM CaCI, ) = 45 ; n ("I mM EGTA- 0.6 mM CaCI2) = 48; n (1 mM EGTA-O.4 mM CaCI,) = 57. (C) Western blot analysis of homogenates of cells incubated with different [Ca'+]o for 48 h and probed with anti-TMP4 Abs. The amount of TMP4 diminishes when ce ll s were H grown in media with reduced [Ca ].. levels. As a loading control 12.5 IJ.g of the protein samples were analyzed by SDS-PAGE, followed by si lver staining according to the method of Wray et al. (105).

esis. In contrast, in higher eukaryotes intracellular transport of ing CRC-II si lencing, also entails a reduced number of docked/ newly formed secretory organelles has been described as being dischargeable trichocysts. insensitive to [Ca2 +] (68). From this one may conclude that the In Paramecium, just as in other secretory systems, a cytosolic reduced number of total trichocysts may be mainly due to Ca2+ signal is required for membrane fusion (24, 47), while inhibited biogenesis which, with ongoing division activity dur- exogenous Ca2 + is required for contents release (10). Dense

A ..2 1.4 ;;:: AED 4-CmC caffeine

1.3

1.2

1.1

1.0 ~.A_' " B

time (s) time (s) time (s) o 10 20 30 40 o 10 20 30 40 o 10 20 30 40 - pPD-C1C2; n =5 , bars =s . e. m. ----- pPD-GFP; n = 5, bars =s . e. m. FIG. 12. Knockdown of CRC-IV-l channels impairs CaH release from cortical stores. Time course of rCa2+]; changes in cortical regions of Fura Red-injected cells, silenced in CRC-IV-l receptors (red), or mock-treated cells (pPD-GFP, black), fo ll owing stimulation with AED or the ryanodine receptor agonists, 4-CmC and caffein e. Stimulation was performed at low [Call L by adding 1 mM BAPTA to yield [Ca' l.. - 30 nM H (A) or under standard conditions ([Ca ]., - 100 IJ.M) (B). 3618

core-secretOlY vesicles in metazoans usually contain a rather data are also supported by electrophysiological recordings high calcium content, e.g., tens of millimolar (15, 67), but from lipid-reconstituted P. tetraurelia cortex fragments (108) 2 organell ar Ca + is not detectable in trichocysts by X-ray mi­ whereby several channel functions have been detected, one of croanalysis, in contrast to that in alveolar sacs of Paramecium them with properties of an organell ar Ca 2 + -release channel of 2 (33). Only access of extracellular Ca + to trichocyst contents the type we describe here. via the fu sion pore can cause their "decondensation" (sever­ Diversification of Paramecium CRCs. The presence of 34 alfo ld stretching upon vigorous exocytotic discharge) (10). This proteins di stantly related to IP3Rs and RyRs in the Parame­ capability is retained by the residual tri chocysts remaining aft er cium genome reflects a wide expansion of such channels com­ CRC-II silencing, while de novo fo rm ation is inhibited. pared to the situatio n in metazoans (see "Indroduction") and, In essence, th e Ca2 + -based phenomena outlined above can as known thus far, in other unicellular systems (4, 13, 96) (Fig . explain reduced trichocyst biogenesis and consequently re­ .L and 13). A simil arl y broad diversifi cation has been observed duced docking after CRC-IJ-l silencing. A ll of these mecha­ also within many other Paramecium gene families (32, 84, 86, nisms await scrutinized investiga tion. Another salient point of 101), which can be explained as fo ll ows. Owing to a recent our work, however, is that both fa milies of P. tetrauretia CRCs whole-genome duplication, Paramecium genes often occur as analyzed exert wid ely diffe rent functions in the secretory cycle pairs of closely related paralogues (5), which is the case with 20 of Paramecium. genes of the 34 CRCs described here. Expansions within gene Functional assignment of CRC-IV-! channels to alveolar families in Paramecium may compensate for the absence of sacs. We currently fi nd CRC-IV-l illl mun olabelin g on alveolar alternative mRNA splicing (40), a pathway known to ensure sacs and in the ER. How does that compare to the previous diversifi cati on of mamm ali an 1P3Rs and RyRs (9, 28). We 2 findin g that th e Ca + signals required for stimulated exocytosis assume a similar situation for Tetrahymena, which according to would originate primarily fro m alveolar sacs (75)? Clearly, the the TransportDB (http://www.membranetransport.org [83]) considerable identity (nearly 85%) of the ORFs of both CRC­ possesses 23 CRCs. Thus, expansion of such channel types IV-J genes would entail that sil encing would affect both iso­ seems to be a signifi cant fea ture or cili ates. forms, since diffe rential downregul ation would require > 15 % The Paramecium chann els we descri be as buna fid e CR Cs difference in identity (85). Close similarity al so restrains us (Fig. 1) represent novel types, with the exception of the re­ from any diffe rential Ab labeling, whereas expression as OFP cently described CRC-II-l channels, which possess typical fea­ fusion proteins is hampered by the mere size of the CRC tures of IP3Rs (53). The other P. tetraurelia CR Cs displ ay molecules. unusual combinations of one or several characteristics of

Bearing in mind the considerable ambiguity of pharmaco­ IP3Rs, of RyRs, of both, or of unrelated parts. Figure lA logical tools in work with ciliated protozoans (77), we rather presents the like ly relationship between the P. tetraurelia reanalyzed the effects of different trigger agents previously CRCs, based on ORF analysis. (Note that RyRs have not been used, among them polyamines (49, 74, 78) and the permeable included in Fig. l A because, due to their size, only conserved RyR agonists, 4-CmC and caffeine, which all normally produce regions of the channel domains are considered in alignments, 2 trichocyst exocytosis in parall el to SOC-type Ca + signaling which would lead to cluster formation with mamma lian IP3 Rs. ) ( 46-48). It appears that precursor channels would have undergone in­ We now obse rve by flu orochro me analys is after CR C-IV-l creasin g di ve rsifi cation to serve th e multiple speciali zed fun c­ silencing that, with all of three trigger agents, exocytosis is ti ons of Ca2 + signals in Paramecium (see the introduction) . In inhibited in parallel to a reduction of the Ca2 + release from contras t to th e most strin gently defin ed CRC-TI - I chann els alveolar sacs. In conjunction wi th the subcellular localizati on (53), CRC-IV-l channels di splay some aberrant features, as of CRC-IV-l to alv eolar sacs, the generation of Ca2+ signals, outlined in "molecular characterization of CRC-IV-l chan­ therefore, supports the assumption that CR C-IV-l channels nels." T he other subfa milies remain to be characterized by 2 are the Ca + -release channels relevant for trichocyst exocy­ deta iled domain analysis and functional assays, a topic for tosis. future work. Despite the insensitivi ty of CRCs in alveolar sacs, now iden­ Evolutionary aspects. Currently, Ca2+ signaling in unicellu­ ti fied as CRC-JV-l chann els, to rya nodin e (54), their sensitivity lar eukaryotes meets consid erable interest. In the closely re­ to 4-CmC (46) and caffein e (48) clearly argues for RyR-related lated parasites Plasmodium and Toxoplasma (both being mem­ channels, since this is characteristic of RyRs in higher eukary­ bers of the phylum A lveolata , just like ciliates), Ca2 + signaling otic systems (22, 26, 39). Unfortunately, no binding domains is mandatory for host cell penetration (17). However, it is not are known for caffeine, wh ich also requires tens of millimolar yet known whether the "inner membrane complexes" (alveolar 2 concentrati ons in other cell s, just as in Paramecium (25, 48). sac-li ke structures) play a role in Ca + signaling (16). IP3 Rs Putative ryanodine-binding sites normally present in the last are assumed to occur, although other types-yet to be identi­ C-terminal transmembrane domain (100) are not present in fied- have also been envisagcd (57, 65). In sum, clear molec­ the primaly sequence of P. tetraurelia CRC-IV-l (Fig. 2D), ul ar identificat ion of CR Cs in th ese organisms has no t been thus explaining their insensitivity. 4-CmC binding sites in RyRs possible thus far, despite consid erabl e efforts (66). A similar are also well characterized in higher eukaryotes (26, 27). Com­ situation occurs in the unicellul ar fungi, Candida albicans and 2 pari son with Paramecium sequences reveals that this site is Neurmpora crassa . For both species IP3-dependent Ca I sig­ conserved in CRC-IV-l channels (see Fig. S2 in the supple­ naling is evi dent (14, 87) (see the in troduction), but no IP3 R mental material) . Their insensitivity to ryanodine, therefore, orthologues show up in genomi c screens (note that for all of recalls the situation in malignant hyperthermia pati ents whose these organisms complete genome information exi sts). This mutated RyRs are sensitive selective ly to 4-CmC (102). Our suggests th at evolution has led to di ve rsifica ti on to an ex tend 3619

IP -blndlnQ channel A B MmlP R1 (2749aa) MmlP3R1 (a) 224 104 2278 2589 3 AplP3R (b) DmlP3R (e) AplP3R (2698aa) 100 BmlP3R ] (d) CeITR-1 DmlP3R (2833aa) CeITR-1 (2846aa) 100 Mb C 180082 ] BmlP3R (2767aa) L- Mb C_13000054 (e) £===-, Mb C_180082 (2669aa) lI100 Mb C_50131 Mb C_50131 (2571 aa) PtlP3RN1 Mb C_13000054 (2614aa) 100 PtlP3RN2 39 PtlP3RN1 (2890aa) r---- TC00997500 PtlP3RN2 (2888aa) '----- TC00545830 99 (f) TC00997500 (3133aa) 100 PtCRC-IV-1a TC00545830 (3387aa) r------"'O<.j PtCRC-IV-1 b PtCRC·IV·1a (2997aa) 53 r------TC00138560 PtCRC-IV·1 b (3000aa) '------TC00762860 Tt_00138560(3006aa) r------Mb C_37000059 (e) TC00762860 (2887aa) '------DdiplA (9) Mb C_37000059 (2869aa) DdiplA (3177aa) 100 Lm XP _001682106 ] Li XP _0014644518 (h) Lm XP _001682106 (2874aa) LIXP_0014644518 (2874aa) L--- == Tb tba1.306.m00053 Tb tba1 .306.m00053 (3099aa) 1OiL100 Tc tea1.81 06.m00009 Tc tca1.8106.m00009 (3011aa) '------PC01069 (I) PC01069 (2916aa) ,...------C,_159958 (j) C,_159958 (3140aa) 49 AaJ2654 (k) Aa_72654 (5282aa) ~ -.------~------~--- FIG. 13. IP,R-related pro tozoan sequences. (A) Neighbor·joining tree (with 1,000 bootstrap replicates) of phyl ogenetic relationships between

IP3R·related orthologues. Note that systematic groups are designated by (a to k). For deuterostomes (a and b) , we included sequences representing IP3Rs from the mammalian M. mLisculLis (MmIP3Rl) (a) and fro m the echinoderm As/erina pectini/era (b). For protostomes, we included IP3Rs from D. melanogaster (c) and from the nematodes C. elegans and BrLlgia malayi (d). Among unicellular organisms orthologues are present in the choanofl agell ate Monosiga brevicoflis (e) and in the ci liates P. te/raurefia and T. /hermophila (f). IP3 R-related genes also exist in Phy/ophthora in/es/an s (i), the green algae Chlamydomonas reinhard/ii (j), the chlysophycean algae ALireococcLis anophagejj,'erens (k). and the ipfA gene of Dictyos/eliwn discoideum (g) (96) . (h) Among protozoan parasites, IP3R orthologues are present in trypanosomes such as Leishmania major, Leishmania in/antum, Tlypanosoma brucei, and Tl ypaJlOsoma crLl zi. Seq ue nces we re identified either by BLAST£> searches of respective databases or by using the TransportDB (http://www.membranetransport.org [83]). Accession numbers of the sequences are summarized in T a ble S4 in the supplemental material. Bootstrap support va lues for the nodes are in dicated, as are evolutionary distances by the scale bar, below. (B) Sequence analysis of IP3 R-related protozoan sequences. Conselved regions were determined by using the BLASTP 2.2 .1 9 program (http://www.ncbi .nlm .nih .gov [3]) with the same proteins as those presented in pa nel A, ali gned with M. mLlseullls IP3Rl as query sequence. Alignme nts are color coded by score with five score ranges. T hc positions of nanking residues and correspondi ng e-valu cs are summari zed in Table S4 in th e suppleme ntal material.

that the perceptibility of IP3R-related sequences may be re­ parts of orthologues from protozoa are mainly pronounced in stricted to a limited number of unicellular species. regions of. the C-termin al channel domain (Fig. DB; for de­ According to the TransportDB (83), which allows genome­ tails, see F ig. S5 and Table S4 in the supplemental material) . wid e comparison of predicted membrane transporters across a An exceptional position holds true for CRCs from M. brevicol­ broad range of organisms, IP3R-related genes are present in lis (13) with clearly recognizable similarities to both, ci li ate several unicellular organisms (Fig. 13). In addition, to ciliate CRCs and mammalian IP3Rs (Fig. 13). The recent identifi ca­ CRCs, they exist in the algae Chlamydomonas reinhard/ii and tion of such channels in M . brevicollis, although not yet ana­

Aureococcus anophagejJerens (note that they are absent from lyzed in any detail , suggests an IP3R-mediated mechanism in higher pl ants [103, 106]) and in several Phy/ophthora species. choanoft agell ates (13). Their comparatively hi gh degree of Among protozoan parasites, IP 3R-related genes occur in conservation correlates with the evolutionary position of these Tlypanosoma and Leishman.ia species. There exist four genes organisms as putative precursors of metazoans (44, 60). Con­ coding for IP3Rs (13) in lhc choanonagcll atc Monosiga brevi­ comitantly, recent genomic analysis of the M. brevicollis ge­ collis, wh ich were identifi ed in a database screen of th e recently nome (44) has suggested that complex Ca2 + signaling may unraveled genome (http://genome.jgi-psf.orgIMonbrl[44]),as have evolved early on (13) . we ll as the IP1' receptor-li ke protein JplA (96) from Dic/yosle­ The IP3 type of signal transduction is clearly present already lhun discoideum. in Paramecium (53). Beyond th al, wc now find stringe nt evi­ In fact, such sequences diverge from metazoan IP3Rs to a dence of additional CRCs, including the novel CRC-IV-l similar degree as observed for Paramecium CRCs (Fig. 13). channels. These may belong to an evolutionarily old type of Alignments with Mus musculus IP ~ Rl reveal, that conserved signal transduction pathway. Taken the number of subfam ili es 3620

of P. tetraurelia CRCs togeth er, CRCs may have di ve rsifi ed 19. Claros, M. G., and G. von Heijne. 1994. TopPred II: an improved software early on in evolution to an unexpected extent, involving not for memhrane protcin structurc prcdicti ons. Com put. Appl. Biosci. 10:685- 686. only 1P3Rs and RyR types, but additional ones still to be 20. Deprez, 1'., M. Gautschi, and A. Helcnius. 2005. More tha n one glycan is characteri zed in detail. needed for ER glu cosidase II to allow entry of glycoprotcins into the calnexin/calreti culin cycle. Mol. Cell 19:'183-195. 2 1. Dessen, P., M. Zagulski , R. Gromadka, H. Pla ttner, R. Kissmehl, E. Meye r, ACKNOWLEDGMENTS M. Betermier, J. I':. Schultz, J. U. Linder, R. E. Pearlma n, C. Kung, J . Forney, B. H. Satir, J . L. Van Houten, A. M. Keller, M. Froissard, L. We thank l ean Co hen and Linda Sperling (CNRS, Gif-Sur-Yvette, Spcrling, and J. Cohen. 200'l. Parameciulll genome survey: a pil ot project. France) for enabling us to perform the screening experiments and Trends Genet. 17:306- 308. additionally Genoscope (Evry, France) for access to the setver with the 22. Ehrlich, B. 1':., E. Kartan, S. Bezprozvannnya, and 1. Bezpr ozvanny. 1994. Paramecium sequencing data. We th ank especially Linda Sperling for T he pharmacology of intracellular Ca2+ -release channcls. T rends Pharma­ providing th e Abs to TMPl and TMP4, as well as for help in organi zing col. Sci. 15: 145-149. accession numbers from the Paramecium database. We are grateful to 23. Eisen, J . A., R. S. Coyne, M. Wu, D. Wu, M. Thiagarlljan, J. R. Wortman, J . H. Badger, Q. Ren, P. Allledeo, K. M. Jones, L. J . Tallon, A. L. Deicher, Marek Zagulsky (Polish Academy of Science, Warsaw, Poland) for S. L. Salzberg, J. C. Silva, B. .I. Haas, W. H. Majoros, M . Farzad, J. M. initial help with sequencing at an early stage of this work. We also Carlton, R. K. Smith, Jr., .I. Garg, R. I':. Pearllllan, K. M. Karrer, L. SlIn, thank Thomas Wassmer and Roland Kissmehl for helpful discussions, G. Manning, N. C. Elde, A. 1'. Turkcwitz, D . .I. Asai, D. I':. Wilkes, Y. Wang, Deisy Geisinger for providing the POI expression plasmid, and Lau­ H. Cai, K. Collins, B. A. Stewart, S. R. Lce, K. Wilamowska, Z. Weinberg, retta Nejedli for technical assistance (all at the University of Con­ W. L. Ruzzo, D. Wloga, J. Gaertig,.I. Frankel, C. C. Tsao, M. A. Gorovsky , stance, Constance, Germany). In addition, we thank E. May for use of P. J. Keeling, R. F. Wallcr, N. J . Patron, J . M. Cherry, N. A. Stover, C. J. the LSM510 facilities and A. Burkle fo r access to the Bio-Rad iCycier. Krieger, T. C. del, H. F. Ryder, S. C. Willialllson, R. A. Barbeau, I':. P. This study has been supported by the Deutsche Forschungsgemein­ Hamilton, and I':. Orias. 2006. Maeronuclear genome seque ncc of the ciliate Tetrahymefla th em lOphila, a model e ukaryote. I'LoS . BioI. 4:e286. schaft (grants to H.P., including project C4 of TR-SFBll). 24. Erxleben, C., N. Klauke, M. Flotenmeycr, M. P. Blanchard, C. Braun, and H. Plattner. 1997. Microdomain Ca2+ acti vati o n during exoeytosis in Par­ R EFERENCES amecium cell s. Supcrpositio n of local subplasmalemmal calci um store ac­ I. Ahluwalia, J. P., J. D. Topp, K. Weir"ther, M. Zimmerman, and M. ti vation by local Ca2+ in nux. J. Cell 13iol. 136:597- 607. Stamnes. 2001. A role for calcium in stabilizin g transporl vesicle coats. 25. Erxleben, C., and H. Plattner. 1994. Ca2 ~ release from subplasmale mm al J. Bio I. Chem. 276:34148- 34155. sto res as a primary evcnt du ring exocytosis in Paramecium ce ll s. J. Cell BioI. 2. AII~n , R. D., and Y. Naitoh. 2002. Osmo regulatio n and contractile vacuoles 127:935-945. of protozoa. Inl. R. Cytol. Survey Cell BioI. 215:35 1-394. 26. Fessenden, J. D., W. Feng, I. N. I'essah, and P. D. Allen. 2006. Amino acid 3. Altschul, S. F., T. L. Madden, A. A. SchutTer, J. Zhang, Z. Zhang, W. Miller, residues Gln4020 and Lys4021 of the ryanodine recepto r type 'I are re­ lind D. J. Lipman. 1997. Gappcd BLAST and PSI-BLAST: a nCw genera­ quired for activation by 4-chl oro-m-crcsol. J. Bio I. Chern . 281:21022- 2103 1. ti o n of protein database search programs. Nucleic Acids Rcs. 25:3389- 3402. 27. Fessenden, J. D., C. F. Perez, S. Goth, 1. N. Pessah, and 1'. D. Allen. 2003. 4. Arnaiz, 0., S. Cain, J. Cohen, and L. Sperling. 2007. ParamcciumOB: a Identilicati on of a key ueterminant of ryanouine recept or type 1 required community resource th at integrat es th e Paramecium telraurelia genome fo r activati on by 4-chl oro·m-cresol. J. BioI. Chern . 278:28727-28735. scquence with genctic data . Nucleic Acids Res. 35:0439- 0 444. 28. Fill, M., and J . A. Copello. 2002. Rya nodine receptor calci um rclease 5. Aury, J. M., O. Jaillon, L. Duret, B. Noel, C. Jubin, B. M. Porcel, B. channels. Phys iol. Rev. 82:893- 922. Segurens, V. Daubin, V. Anthouard, N. Aiach, O. Arnaiz, A. Billaut, J. 29. Fleischer, S. 2008. Personal recollectio ns on the discovery of the ryanodine Beisson, I. Blanc, K. Bouhouche, F. Camara, S. Duharcourt, R. Guigo, D. receptors of muscle. Biochem. Biophys. Res. Commun. 369:195- 207. Gogendeau, M. Katinka, A. M. Keller, R. Ki ssmehl, C. Klotz, F. KolI, M. A. 30. Galvani, A., and L. Sperling. 2002. RNA interfcrence by feeding in Para ­ I.e, G. Lepere, S. Malinsky, M. Nowacki, J . K. Nowak, H. Plattner, J. mecillm . Trends Genet. 18:11 - 12. Poulain, F. Ruiz, V. Serrano, M. Zaglliski, P. Dessen, M. Betermier, J. 3 1. Gautier, M. C., L. Sperling, and L. Madeddu. 1996. Cloning and scqucnce Weissenbach, C. Scarpelli, V. Schachter, L. Sperling, Eo Meyer, J. Cohen, analys is of genes coding for paramecium secretory granule (trichocyst) and P. Wincker. 2006. Global trends of wh ole-genome duplicati ons rc­ proteins: a unique protein fold for a family of polypeptides with ditTerent vealed by the cili ate Pliram ecium tetral/refill . Nature 444:1 7 1- 178. primary structurcs. J. BioI. Chcm. 271:10247- 10255. 6. Beckers, C. J., and W. E. Balch. 1989. Cldcium and GTP: essential com­ 32. Gogendeau, D., C. Klolz, O. Amniz, A. Malinowska, M. Dadle" N. G. de ponent s ill ves icul ar trnfTi ckin g het\\'ee ll th e endopl asmi c reticululll and Loubresse, F. Ruiz, F. KolI, lind J. Beisson. 200H. Fun ctional di ve rsificati on Golgi apparatus. J. Ccll BioI. 108: 1245- 1256. of centrins and cell morphological complex ity. J. Cell Sci. 121 :65-74. 7. Berddge, M. J., M. D. Bootman, and H. L. Rodedck. 2003. Calcium sig­ 33. Hardt, M., and H. Plattner. 2000. Sub-second qucnched-ll ow!X-ray mi cro­ nalling: dynamics, homcostasis and remodeling. Nat. Rev. Mol. Cell Bi oI. analysis shows rapid Ca2+ mobilization from cortical stores parallelcd by 4:5 17- 529. Ca 2+ innux during sy nchro nous cxocy LO sis in Pa ram ecium cel ls. E ur. J. Ce ll 8. Berridge, M . .I., P. Lipp, and M. D. Bootman. 2000. The versatility and Bio I. 79:642-652. unive rsality of calci um signalling. Nat. Rev. Mol. Cell BioI. 1: 11 - 2 1. 34. Harullloto, T., and A. Miyake. 199 1. Oefcnsive functi on o f trichocysts in 9. Bezprozvanny, I. 2005. The inosit ol I ,4,5-trisphosphate reccptors. Cell Cal­ Pall/mecilllll. J. Exp. Zool. 260:84-92. cium 38:261- 272. 35. Hauser, K. , W. J. Haynes, C. Kung, H. Plattner, a nd R. Kissmeh!. 2000. 10. Bilinski , M., H. Plattner, and H. Matt. 198 1. Sccretory pro tein deconden­ Express ion of the green lIuo rescenl prol c.::.i n in Paramecium lelrtl Ilrelia . Eur. sation as a distin ct, Ca 21 -media ted t;vc nt dllring th e fin al sleps uf exucy tos is J. Cell BioI. 79:1 44- 149. in Pliramecil/m ccll s. J. Cell Bio I. 88: 179-188. 36. Hauser, K., N. Pavlovic, R. Kissmehl, and H. Plattner. 1998. Molecul ar II . Boehning, D., D. O. Mak, .I . K. Foskett, and S. K. Joseph. 200 I. Molecul ar characte ri zati o n of a sareo(endo)plasmi c reticulum Ca2+ -AT Pase gcne determinants of io n perm eati on and sclccti vity in in ositol 1,4,5-tri sphos­ from Para mecium lelraurelia and loca li za tion of its gene prod uct to sub­ phate receptor Ca2 + channels. J. Bio I. Chern . 276:13509- 135 12. plasmalemm al calci um stores. Biochem. J. 334:3 1- 38. 12. Browning, J . L., and D. L. Nelson. '1976. Biochemical studics of the exci t­ 37. Hauser, K., N. Pavlovic, N. Klauke, D. GeiSSinger, and H. Plattner. 2000. 15 2 abl e membrane of Param ecium aurelia. I. 1 Ca + flu xes ac ross res ting and G reen flu orescent protei n-tagged sarco(endo)plasmic reti culum Ca 2 ~ ­ excited membranc. Biochim. Bio phys. Acta 448:338-35'l. AT Pase overexprcssion in Paramecillm cells: isoforms, subce llular locali za­ 13. Cai, X. 2008. Unicellular Ca' + signaling 'toolkit' at the o ri gin of mctazoa. ti on, biogcnesis of cortical calcium sto res and fun ctional aspects. Mo l. Mol. BioI. Evol. 25: I357 - 'I36'l. Mi crohi ol. 37:773- 787. 14. Calvert, C. M., and D. Sanders. 1995. Inositol trisphosphate-dependent and 38. Helenius, A. , and M. Aebi. 2004. Roles of N-linked glyca ns in thc e nd o­ -i ndependent Ca" mobilizati o n pathways at thc vacuolar membra ne of plasmi c reticulum. Annu. R ev. Biochem. 73: 101 9-1049. Clilldida albicalls. J. BioI. Chcm. 270:7272-7280. 39. Herrma nn-Frank, A., M. nichter, S. Sarkozi, U. Mohr, and F. Lehmann­ 15. Camacho, M., J. D. Machlldo, J. Alv.. rez, and R. Borges. 2008. Intravesicu­ Horn. 1996. 4-Chl o ro-lIl-cresol, a po tent and spccific acti vator of thc skel­ lar calci um release mediates the motio n and cxocytosis o r secretory o r­ ctal muscle ryanodine recepto r. Biochim. Bio phys. Acta 1289:31-40. ganell cs: a study with adrenal chrolll airill cell s. J. Bio I. e hem. 283:22383- 40. Jaillon, 0., K. Bouhouchc, J. F. Gout, .I. M. Allry, B. Noel, B. Saudemont, 22389. M. Nowacki, V. Serrano, B. M. I'orcel, B. Segurens, A. I.e Mouel, G. Lepere, 16. Carruthers, V. , and J. C. Boothroyd. 2007. Pulling together: an in tcgrated V. Schachter, M. Betermier, .I . Cohen, P. Wincker, L. Sperling, L. Duret, model of Toxop lasma ccll in vasio n. Curr. Opin . Microbiol. 10:83- 89. and E. Meyer. 2008. T ranslati o nal cont rol of intron splicing in e ukaryotcs. 17. Carruthers, V. B., and L. D. Sibley. 1999. Mobili zation of intracellul ar Nature 451:359- U I5. calcium stimulates mi croneme dischargc in Toxoplasma gOlldii. Mol. Mi ­ 4 1. Kaneshiro, E. S., L. S. Beischel, S . .I . Merkel, and D. I':. Rhoads. 1979. The cro biol. 31:421-428. fatty- acid co mpos iti on of ParameciulIl {wrelia ce lls and cilia - changes with 18. Cia phalli, D. E. 2007. Cal cium signalin g. Cell 131 :'1047- 1058. cul ture age. J. Protozool. 26: 147- 158. 3621

42. Kell er, A. M., and .I . Cohen. 2000. An indexed genomic li brary for Pllra­ 67. Nlcaise, G., K. Maggio, S. Thirion, M. Horoyan, and E. Keicher'. 1992. The mecitllll compl ementa tio n cloning. J. Eukaryol. Microbiol. 47:'1- 6. calcium loading o f secretory granules: a possible key event in stimulus­ 43. Korboeur, D., and .l . Cohen. 1990. A Ca2+ innux associated with exocytosis secreti o n coupling. Bio I. Cell 75:89- 99. is spccifi ca ll y abo lishccl in a PII/'tlm ecilll1l exocytoti c mutanl. J. Cell Bio I. 68. Nishida, K., S. Yamasaki, Y. Ito, K. Kabll, K. Hattori, T. Tezllka, H. 111 :2527-2535. Nishizumi, D. Kitamura, R. Goitsuka, R. S. Geha, T. Yamamoto, T. Yagi, 44. King, N., M. J . Westbrook, S. L. Young, A. Kuo, M. Abedin, J . Chapma n, and T. Hirano. 2005. FCERI-mediated mast cell degranulatio n requires S. Fairclongh, U. Hellstcn, Y. Isogai, I. Letunic, M. Marl', D. Pincns, N. ca lcium-independent microtubul e-depende nt translocation o f granules to Putnam, A. Rokas, K. J . Wright, R. Zuzow, W. Dirks, M. Good, D. Good­ the plasma membrane. J. Cell BioI. 170:115- 126. stein, D. Lemons, W. Li, J. B. Lyons, A. Morris, S. Nichols, D. J . Richter, 69. Passos, A. 1'. D., and C. R. S. Garcia. 1998. Inositol 1,4,5-trisphosphate A. Salamov, J . G. Sequcncing, P. Bork, W. A. Lim, G. Manning, W. T. in duced Ca 2 ~ relcase from ehloroquine-sensitivc and -insensitive intracel­ Miller, W. McGinnis, H. Shapiro, R. 'Ijian, I. V. Grigoriev, and D. Rokhsllr. lul ar stores in the intraerythrocyti e stage of the malaria parasite Plas/1/O ­ 2008. T hc g~ n umc u f the choanunagell atc MOllosigll brevicollis and the dillm C//(/iJlllldi. Biochem. Biophys. Res. Commun. 245:155- 160. o ri gi n of metazoans. Nature 451 :783- 788. 70. Patton, C., S. Thompson, and D. Epel. 2004. Some precautions in using 45. Kissmehl, R., I. M. Sehring, E. Wagner, and H. Plattner. 2004. Immuno­ ehelato rs to buffe r metals in biological solutions. Cell Calcium 35:427-43 1. locali zati o n of actin in PlirallleciulII cell s. J. Histochem. CYlOchem. 52: 1543- 71. Plattner, H. 2002. My favorite cell : Part/l7leciul7I. Bioessays 24:649- 658. 1559. 72. Plattner, H., C. Braun, and .I. Hentschel. 1997. Facilitatio n of membrane 46. Klauke, N., M. Blanchard, and H. Plattner. 2000. Polyamine triggering of fu sion during exocytosis and exocytosis-coupled endocytosis and accelera­ cxocytosis in Parttm edum in vo lves an extrace llul ar Ca2 + /(polyvalcnt ca t­ tion of "ghost" detachm ent in Param ecium by ex tracellular ca lcium. A io n)-sensing receptor, subplasmalemmal Ca-store mobilization and store­ qucnched-now/freeze-fracture analys is. J. Mcmb r. B io I. 158:197-208. operated Ca2+ -influx via unspeci fi c cati on channels. J. Membr. Bio I. 174: 73. Plattner, H., A. Habermann, R. Kissmehl, N. K lauke, I. Maj oul, a nd H. D. 141- 156. Soling. 1997. Differential distribution of calcium stores in Paramecium cell s: 47. K lauke, N., and H. Plattner. 1997. Im aging o f Ca2 + transients induced in occurrence of a subplasmalemmal sto re wit h a calsequestrin-li ke protein. Pliramecium cell s by a polyamine secretagogue. J. Cell Sci. 1l0:975- 983. Eul'. J. Cell BioI. 72:297-306. 2 48. Klauke, N., and H. Plattner. 1998. Caffeine-induced Ca ' transients and 74. Plattner, H., and J . Hentschel. 2006. Sub-second ce ll ular dynamics: time­ exoeytosis in Pli/'tlmeciulIl cells: a correlated Ca2+ imaging and quenehed­ resolved electron mi croscopy and fun ctio nal correlatio n. Inl. Rev. CylOl. lI uw/fr eeze-fracture analys is . .I . Membr. BioI. 161:65- 8 1. 255:1 33- 176. 49. Knoll, G., C. Braun, and H. Plattner. 199 .1 . Quencheci Row analysis of 75. Plattner, H., and N. Klauke. 200 I. Calcium in ciliated protozoa: sources, cxocytosis in ParWnecillnl ce lls: lime course, changes in membrane stru c­ regulation, and calcium-regul ated cell functions. Int. Rev. Cyto l. 201 : 11 5- tUfe, and calcium requ irements revealed after rap id mixing and ra pid freez­ 208. ing o f intact cells. J. Ccll BioI. 113:1 295- 1304. 76. Plattuer, H., K. Reichel, H. Matt, J . Beisson, M. Lefort-Tran, and M. 50. Kumar, S., K. Tamura, and M. Ne i. 2004. MEGA3: integrated soft ware for I'ouphile. 1980. Gene ti c dissecti on of the final exocytosis steps in Pal'lll7l e­ molccular evolutionary gene ti cs analysis and seque nce alignment. Brie f. ciul7I lell'llurelia cell s: cytochemical determination o f CaH -ATPase acti vity Bioinform. 5: 150-"163. over performed exocytosis sites. J. Cell Sci. 46:17-40. 51. Kung, C., and Y. Sairni. 1985. Ca H channels of PlIl'lImecium: a multidisci­ 77. Plattner, H., I. M. Sehring, C. Schilde, and E. M. Ladenbnrger. 2009. plinary study. CUr l'. Top. Membr. Transport 23:45-66. Pharm acology of ci li ated protozoa: drug (in)sensitivity and experimental 52. Kyte, .I., and R. F. Doolittle. 1982. A sim ple method for display ing the drug (ab)use. Int. Rev. Cell Mo l. Bio I. 273:163- 218. hydropathic character of a protein. J. Mo l. BioI. 157:105- 132. 78. Plattner, H., R. Stiirzl, and H. Matt. 1985. Synchronous exocytosis in 53. Ladenburger, E. M., I. Korn, N. Kasielke, T. Wassmer, and H. Plattner. Part/l7l eciUfll cells. IV. Polyamino compounds as potent trigger age nts for 2006. A n Ins(,I,4,5)P, receptor in Paramecium is associ ated with the osmo­ re peatable trigger-redocking cycles. E li I'. J . Cell Bio I. 36:32-37. regulatory system. J. Cell Sci. 119:3705- 37 17. 79. Pollack, S. 1974. Mutations affecting the trichocysts in Pa rame ~iwll aurelia. 54. Lunge, S., N. Klauke, a nd H. Plattner. 1995. Subplasmalemmal CaH sto res I. Morphology and description of the mutants. J. Protozool. 21 :352-362. of probable relevance for exocytosis in Pal'llmecium: alveolar sacs share 80. I'onting, C. 1'. 2000. Novel repeats in ryanodine and II', receptors and some but not all characte risti cs with sarcoplasmic reticulum. Cell Calcium protein O-mannosyltransferases. Trends Bioehem. Sci. 25:48- 50. 17:335- 344. 8"1. l'utney, .I . W., J r. 2007. Recent breakth roughs in the molecular mechanism 55. Livak, K . .I., and T. D. Schmittgen. 200 1. Analysis of relative gene expres­ of capacitative calcium entry (with thoughts o n how we got he re). Cell sion data using real-time quantitative PC R and the 2- "'CT method. Meth­ Calcium 42:103-11 0. ods 25:402-408. 82. Rumoino, 1'., A. Oiaspro, M. Fato, F. Beltrame, and M. Robello. 2000. 56. Lovett, .I. L., N. Marchesini, S. N. Moreno, and L. D. Sibley. 2002. Toxo­ Changes in th e endop lasmic reti culum stru cture of PammecillJ"n primallrelia 2 plasma gondii microncmc secretion in vo lves intracellul ar Ca + release from in relatio n to different cellular physiological states. J. Photoehem. Photo- in osit ol 1,4,5-triphosphate (IPj )/ryanodine-sensitive stores. J. Bio I. Che rn. bio I. B 54:35-42. . 277:25870- 25876. 83. Ren, Q ., K. Chen, and I. T. Paulsen. 2007. TransportDB: a comprehensive 57. Lovett, .I . L., and L. D. Sibley. 2003. Intracellular calcium sto res in Toxo­ database resource for cy topl asmic membrane tr anspor t systems and outer plasma gOlldii govern in vasio n of host cell s. J. Cell Sci. 116:3009-3016. membrane channels. Nucle ic Acids Res. 35:D274-D279. 58. Machemer, H. 1988. Electrophys io logy, p. 185-215. III H.- D. Giirtz (cd.), 84. Ruiz, F., N. Garreall de Loubresse, C. Klotz, J. Beisson, und F. Koll. 2005. Parameci um. Springer-Ve rlag, Berlin, Germany. Centrin deficiency in PlIl'lImeciulIl a ffects the geometry of basal-body du­ 59. Marchesini, N., M. Bolio, G. Hernandez, M. N. Garrido, and E. E. pl icati o n. Curl'. BioI. 15:2097- 2 106. Mnchado-Dolllcnech. 2002. Cellular signalling in Trypallosoma cruzi: bipha­ 85. Ruiz, F., L. Vayssic, C. Klotz, L. Sperling, a nd L. Madeddll. 1998. H omol­ sic behav io ur o f inositol phosphate cycle components evoked by carbachol. ogy-dependent gcne sil encin g in I'il/'llmecillm . Mol. BioI. Cell 9:93 1- 943. Mo l. Bioehem. Parasit o l. J20:83- 91. 86. Sehring, I. M., C. Reiner, .I . Mansfeld, H. Plattner, a nd R. Kissmehl. 2007. 60. Martens, C., K. Vandepoele, and Y. Van de Peer. 2008. Wholc-genome A broad spectrum of actin paralogs in PlIl'lIl'I/eciul1I lelrtlare/ill cells display annlysis reveals molecular innova tions and evol utionary tra nsitions in chro­ differential locali zati on and fu ncti on. J. Cell Sci. 120:177- 190. malveolate species. Proc. Natl. Acad. Sci. USA 105:3427-3432. 87. Silverman-Gavrila, L. B., and R. R. Lew. 2002. An IP, -activated CaH 6 1. Masuda, W., S. Takcnaka, S. Tsuya ma, M. Tokunaga, R. Ynmaji, H. Inui, channc l regulates fungal tip growth. J. Cell Sci. 1l5:5013- 5025. K. Miyatake, and Y. Nakano. 1997. Inosit ol 1,4,5-trisphosphate and cyclic 88. Skollri, F., and J . Cohen. 1997. Gene tic approach to regulated exocylOsis 2 ADP-ribose mobilize Ca ' in a protist, £ lIglellll graci/is. Compo Biochem. using functi onal co mplemcntation in Paflllneciul1l: icicntificaLion oJ th e Physio l. C Pharmaeol. Tox ieol. Endoerino l. 118: 279 -2~3. ND7 genc required for membrane fu sion. Mol. BioI. Cell 8:'1063- 101'1. 62. Michikawa, '1'., H. Hamanaka, H. Otsu, A. Yamamoto, A. Miyawaki, T. 89. Sonneborn, T. M. 1974. PlIrtll'l'lecilll1l ilurelill, p. 469- 594. III C. Kung (cd.), Furuichi, Y. Tashiro, and K. Mikoshiba. 1994. Transmembrane topology Handhook of gene ti cs, vol. 2. Plenum Press, Inc., New Yo rk, NY. and sites of N glycosylat ion of inositol 1,4,5-trisphosp hate recepto r. J. Bio I. 90. Stelly, N., J.P. Mauger, M. Ciaret, and A. Adoutte. 199 1. Cortical alveoli of Chem. 269:91 84-9189. Paramecium: a vast submcmbranous ca lciu m storagc co mpart mcnt. J. Ce ll 63. Mikos hiba, K. 2007. 11'.1 receptor/Ca2 + channel: from discovery to new BioI. 11 3:103- 11 2. signaling concepts. J. Neuroehem. 102: 1426-1446. 9 1. Stock, C., H. K. Grllnlien, R. D. Allen, and Y. Naitoh. 2002. Osmoregul ation 64. Mohamed, I., N. Klallke, J . Hentschel, J . Cohen, a nd H. Plattner. 2002. in PlIl'lIlI1ecillm : in situ ion gradients permit water to cascade through the Functional and nuorochro l11 c analys is of an cxocytot ic mutant yields ev i­ cy tosol to the cont ractile vacuole. J. Cell Sci. 115:2339-2348. dence of sto re-operated CaH innux in Param ecilllll . J. Memhr. Bio I. 187: 92. Tutll sovn, T. A. , and T. L. Mudden. 1999. BLAST 2 Sequences, a new tool 1- 14 . for comparing protein and nucleotide sequences. F EMS Microbia l. Lett. 65 . Nagamllne, K., L. M. Hicks, B. Fux, F. Brossier, E. N. Chini, a nd L. D. 174:247- 250. S ibley. 2008. Abscisic acid controls calcium-dependent egress and develop­ 93. T hiel, G., E. A. MacRobbie, and D. E. Hanke. 1990. Raising the intracellul ar ment in Toxoplasma gOlldii. Nature 451:207- 2 10. level of in osit ol 1,4,5-trisphosphate changes plasma membrane ion trans­ 66. Nugamllne, K., a nd L. D. Sibley. 2006. Comparati ve genomic and phyloge­ port in characean algae. E MBO J. 9:1 737- 1741. netic analyses o f calcium ATPases and calci um-regulated proteins in the 94. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: Apicomplexa. Mo l. BioI. Evo l. 23: 16 13- 1627. im proving the sensitivity of progressive multiple sequence a li gnm ent 3622

through sequence weighting, position-spec ifi c gap pCll alti es, and weight '102. Wehner, M., H. Rueffert, F. Koenig, C. D. Meinecke, a nd D. Olthoff. matrix choice. Nucleic Acids Res. 22:4673-4680. 2003. The lIe2453Thr mutation in the ryanodine receptor gene 1 is 95. Timmons, L., and A. Fire. '1998. Specifi c inte rference by ingested dsRNA. associated with faci lit ated ca lciu m release from sarcoplasmic reticulum Nature 395:854. by 4-ehloro-m-eresol in human myotubes. Cell Calcium 34:163- 168. 96. Traynor, D., J. L. Milne, R. H. Insall, and R. R. Kay. 2000. Ca2+ signalling 103. White, P. J. 2000. Calcium channels in higher plants. Bioehim. Biophys. is not required for chemotaxis in Diclyosielilim. EMBO J. 19:4846-4854. Acta 1465:171 - 189. 97. Tusnady, G. E., and J. Simon. '1998. Principles governing am in o acid com­ 104. Williams, A. J., D. J. West, and R. Sitsapesan. 200'1. Light at the end of position of integral membrane proteins: application to topology prediction. the Ca2 + -release channel tunnel: structures and mechanisms involved in J. Mol. BioI. 283:489- 506. ion translocation in ryanodine receptor channels. Q Rev. Biophys. 34: 98. Vayssic, L., N. Garreau de Loubresse, and L. Sperling. 200 1. Growth and 61 - 104. form of secretory granules involves stepwise assembly but not diU'erential 105. Wray, W., T. Boulikas, V. P. Wray, and R. Hancock. '1981. Silver staining of sorting of a family of secretory proteins in Pam/1/eciulII. J. Cell Sci. 114: proteins in polyacrylamide gels. Anal. Biochem. 118:197-203. 875- 886. 99. Vayssic, L., F. Skouri, L. Sperling, and J. Cohen. 2000. Moiceular genetics 106. Wu, Y., J. Kuzma, E. Marechal, R. Graeff, H. C. Lee, R. Foster, and N. H. Chua. 1997. Abseisie acid signaling through cyclic ADP-ribose in plants. of regulated secretion in Paramecium. Bioehimie 82:269- 288. 100. Wa ng, R., L. Zhang, J. Bolstad, N. Diao, C. Brown, L. Ruest, W. Welch, A. J. Science 278:2126- 2130. Williams, and S. R. Chen. 2003. Residue Gln 4863 within a predicted trans­ 107. Zdobnov, E. M., aud R. Apweiler. 2001. InterProSean: an integration plat­ membrane sequence of the CaH release channel (ryanodine receptor) is form for the signature-recognition methods in InterPro. Bioinformatics critical for ryanodine interaction. J. BioI. Chem. 278:51557-51565. 17:847- 848. 101. Wassmer, 1'., R. Kissmehl, J. Cohen, and H. Plattner. 2006. Seventeen 108. Zhou, X. L., C. W. Chan, Y. Saimi, and C. Kung. 1995. Functional recon­ a-subunit isoforms of Paramecium V-ATPase provide high speciali za tion in stitution of ion channels from Param ecium cortex in to artificial li posOl11cs. localization and function. Mol. BioI. Cell 17:9n- 930. J. Mcmbr. BioI. 144:199- 208.