Development of an expression system in Tetrahymena inner arm dyneins and motile properties of the single-headed subspecies (Dyh8p and Dyh12p).

Diverse inner arm dyneins cooperate with outer arm dyneins to produce ciliary beating. This study demonstrates an expression system for inner arm dyneins in Tetrahymena. The motor domain of inner arm dynein (Dyh8p or Dyh12p) was fused with the tail of outer arm dynein (Dyh3p) and expressed in viable DYH3-knockout (vKO-DYH3) cells. The chimeric dyneins were observed in the oral apparatus and cilia on the cell bodies, and did not change the swimming speed of vKO-DYH3 cells. In a gliding assay, the motor domains of Dyh8p and Dyh12p moved toward the minus ends of microtubules at 0.8 and 0.3 µm/s, respectively. The gliding velocities of Dyh8p and Dyh12p were decreased in 5 mM ATP but not increased in 0.1 or 0.5 mM ADP. This expression system will be useful for molecular studies on diverse inner arm dyneins.

Tracking Mercury in Individual Tetrahymena Using a Capillary Single-Cell Inductively Coupled Plasma Mass Spectrometry Online System.

As a kind of unicellular eukaryotic protozoa, Tetrahymena is located at the bottom of the aquatic food webs and plays an essential role in the bioaccumulation of mercury (Hg). To track Hg in individual Tetrahymena, a capillary single-cell inductively coupled plasma mass spectrometry (ICPMS) online system was developed. The experimental and instrumental conditions were optimized to ensure the signal detected was the Hg uptake in individual Tetrahymena. Moreover, a quantitative method was established and validated by detecting Hg2+ standard solutions. The limit of quantity was calculated to be approximately 3.8 × 10-15 g Hg/cell, and the detection limit for Hg2+ exposure of Tetrahymena was 0.05 µg/L. By using the proposed method, we found the peak became wider with increasing of exposure concentrations, indicating the accumulated Hg by different Tetrahymena varied greatly, and the difference was more significant at higher exposure concentration. This novel method has the advantages of high sensitivity and real-time detection in individual Tetrahymena, and it could be widely used for further tracking the accumulation of mercury and other metals at the single cell level.

LF4/MOK and a CDK-related kinase regulate the number and length of cilia in Tetrahymena.

The length of cilia is controlled by a poorly understood mechanism that involves members of the conserved RCK kinase group, and among them, the LF4/MOK kinases. The multiciliated protist model, Tetrahymena, carries two types of cilia (oral and locomotory) and the length of the locomotory cilia is dependent on their position with the cell. In Tetrahymena, loss of an LF4/MOK ortholog, LF4A, lengthened the locomotory cilia, but also reduced their number. Without LF4A, cilia assembled faster and showed signs of increased intraflagellar transport (IFT). Consistently, overproduced LF4A shortened cilia and downregulated IFT. GFP-tagged LF4A, expressed in the native locus and imaged by total internal reflection microscopy, was enriched at the basal bodies and distributed along the shafts of cilia. Within cilia, most LF4A-GFP particles were immobile and a few either diffused or moved by IFT. We suggest that the distribution of LF4/MOK along the cilium delivers a uniform dose of inhibition to IFT trains that travel from the base to the tip. In a longer cilium, the IFT machinery may experience a higher cumulative dose of inhibition by LF4/MOK. Thus, LF4/MOK activity could be a readout of cilium length that helps to balance the rate of IFT-driven assembly with the rate of disassembly at steady state. We used a forward genetic screen to identify a CDK-related kinase, CDKR1, whose loss-of-function suppressed the shortening of cilia caused by overexpression of LF4A, by reducing its kinase activity. Loss of CDKR1 alone lengthened both the locomotory and oral cilia. CDKR1 resembles other known ciliary CDK-related kinases: LF2 of Chlamydomonas, mammalian CCRK and DYF-18 of C. elegans, in lacking the cyclin-binding motif and acting upstream of RCKs. The new genetic tools we developed here for Tetrahymena have potential for further dissection of the principles of cilia length regulation in multiciliated cells.

Redescription of a Hymenostome Ciliate, Tetrahymena setosa (Protozoa, Ciliophora) Notes on its Molecular Phylogeny.

In recent years, Tetrahymena species have been used as model organisms for research in a wide range of fields, highlighting the need for a fuller understanding of the taxonomy of this group. It is in this context that this paper uses living observation and silver staining methods to investigate the morphology and infraciliature of one Tetrahymena species, T. setosa (Schewiakoff 1892 Verh. Naturh. Med. Ver. Heidelb., 4:544) McCoy (1975) Acta Protozool., 14:253; the senior subjective synonym of T. setifera Holz and Corliss (1956) J. Protozool., 3:112; isolated from a freshwater pond in Harbin, north-eastern China. This organism can be distinguished from other described Tetrahymena species mainly by its single caudal cilium, which is about twice the length of the somatic ciliature. While the Harbin isolate appears similar to the population described by Holz and Corliss (1956) J. Protozool., 3:112, an improved diagnosis for T. setosa is given based on the previous descriptions and the Harbin population. In summary, this species can be recognized mainly by the combination of the following characters: body in vivo approximately 40 µm × 25 µm, 21-26 somatic kineties, one to four contractile vacuole pores associated with meridians 6-11 and a single caudal cilium. The small subunit ribosomal (SSU) rRNA gene and the cox1 gene sequences of Harbin population are also characterized in order to corroborate that the isolated species branches in phylogenetic trees as a T. setosa species. The phylogenetic analysis also indicated that sequences of populations of Tetrahymena species should be published with detailed morphological identifications.

Two Antagonistic Hippo Signaling Circuits Set the Division Plane at the Medial Position in the Ciliate Tetrahymena.

In a single cell, ciliates maintain a complex pattern of cortical organelles that are arranged along the anteroposterior and circumferential axes. The underlying molecular mechanisms of intracellular pattern formation in ciliates are largely unknown. Ciliates divide by tandem duplication, a process that remodels the parental cell into two daughters aligned head-to-tail. In the elo1-1 mutant of Tetrahymena thermophila, the segmentation boundary/division plane forms too close to the posterior end of the parental cell, producing a large anterior and a small posterior daughter cell, respectively. We show that ELO1 encodes a Lats/NDR kinase that marks the posterior segment of the cell cortex, where the division plane does not form in the wild-type. Elo1 acts independently of CdaI, a Hippo/Mst kinase that marks the anterior half of the parental cell, and whose loss shifts the division plane anteriorly. We propose that, in Tetrahymena, two antagonistic Hippo circuits focus the segmentation boundary/division plane at the equatorial position, by excluding divisional morphogenesis from the cortical areas that are too close to cell ends.

Cell Size Control via an Unstable Accumulating Activator and the Phenomenon of Excess Mitotic Delay.

Unstable Accumulating Activator models for cellular size control propose an activator that accumulates in a size-dependent manner and triggers cell cycle progression once it has reached a certain threshold. Having a short half life makes such an activator responsive to changes in cell size and makes specific predictions for how cells respond to perturbation. In particular, it explains the curious phenomenon of excess mitotic delay. Excess mitotic delay, first observed in Tetrahymena in the '50s, is a phenomenon in which a pulse of protein synthesis inhibition causes a delay in mitotic entry that is longer than the pulse and that gets longer the later in the cell cycle the pulse is delivered. The interpretation of this phenomenon championed by Zeuthen and Mitchison in the '60s and '70s is that an unstable activator of mitosis is degraded during the pulse and has to be resynthesized to a threshold level to trigger mitosis; small cells have more time to resynthesize the activator before mitosis and so suffer less excess delay, whereas, large cells have less time thus suffer greater excess delay. Fifty years later, with our detailed understanding of cell cycle biochemistry, we can identify and test candidate Unstable Accumulating Activators. Here I review the field and further develop this concept.

The Green Tetrahymena utriculariae n. sp. (Ciliophora, Oligohymenophorea) with Its Endosymbiotic Algae (Micractinium sp.), Living in Traps of a Carnivorous Aquatic Plant.

The genus Tetrahymena (Ciliophora, Oligohymenophorea) probably represents the best studied ciliate genus. At present, more than forty species have been described. All are colorless, i.e. they do not harbor symbiotic algae, and as aerobes they need at least microaerobic habitats. Here, we present the morphological and molecular description of the first green representative, Tetrahymena utriculariae n. sp., living in symbiosis with endosymbiotic algae identified as Micractinium sp. (Chlorophyta). The full life cycle of the ciliate species is documented, including trophonts and theronts, conjugating cells, resting cysts and dividers. This species has been discovered in an exotic habitat, namely in traps of the carnivorous aquatic plant Utricularia reflexa (originating from Okavango Delta, Botswana). Green ciliates live as commensals of the plant in this anoxic habitat. Ciliates are bacterivorous, however, symbiosis with algae is needed to satisfy cell metabolism but also to gain oxygen from symbionts. When ciliates are cultivated outside their natural habitat under aerobic conditions and fed with saturating bacterial food, they gradually become aposymbiotic. Based on phylogenetic analyses of 18S rRNA and mitochondrial cox1 genes T. utriculariae forms a sister group to Tetrahymena thermophila.

DNA double-strand break formation and repair in Tetrahymena meiosis.

The molecular details of meiotic recombination have been determined for a small number of model organisms. From these studies, a general picture has emerged that shows that most, if not all, recombination is initiated by a DNA double-strand break (DSB) that is repaired in a recombinogenic process using a homologous DNA strand as a template. However, the details of recombination vary between organisms, and it is unknown which variant is representative of evolutionarily primordial meiosis or most prevalent among eukaryotes. To answer these questions and to obtain a better understanding of the range of recombination processes among eukaryotes, it is important to study a variety of different organisms. Here, the ciliate Tetrahymena thermophila is introduced as a versatile meiotic model system, which has the additional bonus of having the largest phylogenetic distance to all of the eukaryotes studied to date. Studying this organism can contribute to our understanding of the conservation and diversification of meiotic recombination processes.

Autophagy prevents autophagic cell death in Tetrahymena in response to oxidative stress.

Autophagy is a major cellular pathway used to degrade long-lived proteins or organelles that may be damaged due to increased reactive oxygen species (ROS) generated by cellular stress. Autophagy typically enhances cell survival, but it may also act to promote cell death under certain conditions. The mechanism underlying this paradox, however, remains unclear. We showed that Tetrahymena cells exerted increased membrane-bound vacuoles characteristic of autophagy followed by autophagic cell death (referred to as cell death with autophagy) after exposure to hydrogen peroxide. Inhibition of autophagy by chloroquine or 3-methyladenine significantly augmented autophagic cell death induced by hydrogen peroxide. Blockage of the mitochondrial electron transport chain or starvation triggered activation of autophagy followed by cell death by inducing the production of ROS due to the loss of mitochondrial membrane potential. This indicated a regulatory role of mitochondrial ROS in programming autophagy and autophagic cell death in Tetrahymena. Importantly, suppression of autophagy enhanced autophagic cell death in Tetrahymena in response to elevated ROS production from starvation, and this was reversed by antioxidants. Therefore, our results suggest that autophagy was activated upon oxidative stress to prevent the initiation of autophagic cell death in Tetrahymena until the accumulation of ROS passed the point of no return, leading to delayed cell death in Tetrahymena.

Biased assembly of the nuclear pore complex is required for somatic and germline nuclear differentiation in Tetrahymena.

Ciliates have two functionally distinct nuclei, a somatic macronucleus (MAC) and a germline micronucleus (MIC) that develop from daughter nuclei of the last postzygotic division (PZD) during the sexual process of conjugation. Understanding this nuclear dimorphism is a central issue in ciliate biology. We show, by live-cell imaging of Tetrahymena, that biased assembly of the nuclear pore complex (NPC) occurs immediately after the last PZD, which generates anterior-posterior polarized nuclei: MAC-specific NPCs assemble in anterior presumptive MACs but not in posterior presumptive MICs. MAC-specific NPC assembly in the anterior nuclei occurs much earlier than transport of Twi1p, which is required for MAC genome rearrangement. Correlative light-electron microscopy shows that addition of new nuclear envelope (NE) precursors occurs through the formation of domains of redundant NE, where the outer double membrane contains the newly assembled NPCs. Nocodazole inhibition of the second PZD results in assembly of MAC-specific NPCs in the division-failed zygotic nuclei, leading to failure of MIC differentiation. Our findings demonstrate that NPC type switching has a crucial role in the establishment of nuclear differentiation in ciliates.

Dynamic Change of Cellular Localization of Microtubule-Organizing Center During Conjugation of Ciliate Tetrahymena thermophila.

To obtain a comprehensive picture of microtubule dynamics during conjugation, the mode of sexual reproduction in ciliates, we combined indirect immunofluorescence and three-dimensional imaging using confocal laser-scanning microscope to visualize the cellular localization of DNA, microtubules, and γ-tubulin, the main component of the microtubule-organizing center in mating Tetrahymena cells. As the conjugational stages proceeded, the distribution of γ-tubulin changed drastically and microtubules showed dynamic appearance and disappearance during meiosis, nuclear selection, nuclear exchange, and the development of new macronuclei. This study highlights the involvement of cytoskeletal regulation in the modulation of germline nuclear motilities required for ciliate reproduction.

Msh4 and Msh5 function in SC-independent chiasma formation during the streamlined meiosis of Tetrahymena.

ZMM proteins have been defined in budding yeast as factors that are collectively involved in the formation of interfering crossovers (COs) and synaptonemal complexes (SCs), and they are a hallmark of the predominant meiotic recombination pathway of most organisms. In addition to this so-called class I CO pathway, a minority of crossovers are formed by a class II pathway, which involves the Mus81-Mms4 endonuclease complex. This is the only CO pathway in the SC-less meiosis of the fission yeast. ZMM proteins (including SC components) were always found to be co-occurring and hence have been regarded as functionally linked. Like the fission yeast, the protist Tetrahymena thermophila does not possess a SC, and its COs are dependent on Mus81-Mms4. Here we show that the ZMM proteins Msh4 and Msh5 are required for normal chiasma formation, and we propose that they have a pro-CO function outside a canonical class I pathway in Tetrahymena. Thus, the two-pathway model is not tenable as a general rule.

Abandoning sex: multiple origins of asexuality in the ciliate Tetrahymena.

BACKGROUND: By segregating somatic and germinal functions into large, compound macronuclei and small diploid micronuclei, respectively, ciliates can explore sexuality in ways other eukaryotes cannot. Sex, for instance, is not for reproduction but for nuclear replacement in the two cells temporarily joined in conjugation. With equal contributions from both conjugants, there is no cost of sex which theory predicts should favor asexuality. Yet ciliate asexuality is rare. The exceptional Tetrahymena has abandoned sex through loss of the micronucleus; its amicronucleates are abundant in nature where they reproduce by binary fission but never form conjugating pairs. A possible reason for their abundance is that the Tetrahymena macronucleus does not accumulate mutations as proposed by Muller's ratchet. As such, Tetrahymena amicronucleates have the potential to be very old. This study used cytochrome oxidase-1 barcodes to determine the phylogenetic origin and relative age of amicronucleates isolated from nature. RESULTS: Amicronucleates constituted 25% of Tetrahymena-like wild isolates. Of the 244 amicronucleates examined for cox1 barcodes, 237 belonged to Tetrahymena, seven to other genera. Sixty percent originated from 12 named species or barcoded strains, including the model Tetrahymena thermophila, while the remaining 40% represent 19 putative new species, eight of which have micronucleate counterparts and 11 of which are known only as amicronucleates. In some instances, cox1 haplotypes were shared among micronucleate and amicronucleates collected from the same source. Phylogenetic analysis showed that most amicronucleates belong to the "borealis" clade in which mating type is determined by gene rearrangement. Some amicronucleate species were clustered on the SSU phylogenetic tree and had longer branch lengths, indicating more ancient origin. CONCLUSIONS: Naturally occurring Tetrahymena amicronucleates have multiple origins, arising from numerous species. Likely many more new species remain to be discovered. Shared haplotypes indicate that some are of contemporary origin, while phylogeny indicates that others may be millions of years old. The apparent success of amicronucleate Tetrahymena may be because macronuclear assortment and recombination allow them to avoid Muller's ratchet, incorporate beneficial mutations, and evolve independently of sex. The inability of amicronucleates to mate may be the result of error(s) in mating type gene rearrangement.

A single cohesin complex performs mitotic and meiotic functions in the protist tetrahymena.

The cohesion of sister chromatids in the interval between chromosome replication and anaphase is important for preventing the precocious separation, and hence nondisjunction, of chromatids. Cohesion is accomplished by a ring-shaped protein complex, cohesin; and its release at anaphase occurs when separase cleaves the complex's α-kleisin subunit. Cohesin has additional roles in facilitating DNA damage repair from the sister chromatid and in regulating gene expression. We tested the universality of the present model of cohesion by studying cohesin in the evolutionarily distant protist Tetrahymena thermophila. Localization of tagged cohesin components Smc1p and Rec8p (the α-kleisin) showed that cohesin is abundant in mitotic and meiotic nuclei. RNAi knockdown experiments demonstrated that cohesin is crucial for normal chromosome segregation and meiotic DSB repair. Unexpectedly, cohesin does not detach from chromosome arms in anaphase, yet chromosome segregation depends on the activity of separase (Esp1p). When Esp1p is depleted by RNAi, chromosomes become polytenic as they undergo multiple rounds of replication, but fail to separate. The cohesion of such bundles of numerous chromatids suggests that chromatids may be connected by factors in addition to topological linkage by cohesin rings. Although cohesin is not detected in transcriptionally active somatic nuclei, its loss causes a slight defect in their amitotic division. Notably, Tetrahymena uses a single version of α-kleisin for both mitosis and meiosis. Therefore, we propose that the differentiation of mitotic and meiotic cohesins found in most other model systems is not due to the need of a specialized meiotic cohesin, but due to additional roles of mitotic cohesin.

Electrophysiological properties of the microstome and macrostome morph of the polymorphic ciliate Tetrahymena vorax.

Polymorphic ciliates, like Tetrahymena vorax, optimize food utilization by altering between different body shapes and behaviours. Microstome T. vorax feeds on bacteria, organic particles, and solutes, whereas the larger macrostome cells are predators consuming other ciliates. We have used current clamp and discontinuous single electrode voltage clamp to compare electrophysiological properties of these morphs. The resting membrane potential was approximately -30 mV in both morphs. The input resistance and capacitance of microstomes were approximately 350 MΩ and 105 pF, whereas the corresponding values for the macrostomes were 210 MΩ and 230 pF, reflecting the larger cell size. Depolarizing current injections elicited regenerative Ca(2+) spikes with a maximum rate of rise of 7.5 Vs(-1) in microstome and 4.7 Vs(-1) in macrostome cells. Depolarizing voltage steps from a holding potential of -40 mV induced an inward Ca(2+) -current (I(ca) ) peaking at -10 mV, reaching approximately the same value in microstome (-1.4 nA) and macrostome cells (-1.2 nA). Because the number of ciliary rows is the same in microstome and macrostome cells, the similar size of I(Ca) in these morphs supports the notion that the voltage-gated Ca(2+) channels in ciliates are located in the ciliary membrane. In both morphs, hyperpolarizing voltage steps revealed inward membrane rectification that persisted in Na(+) -free solution and was only partially inhibited by extracellular Cs(+) . The inward rectification was completely blocked by replacing Ca(2+) with Co(2+) or Ba(2+) in the recording solution, and is probably due to Ca(2+) -activated inward K(+) current secondary to Ca(2+) influx through channels activated by hyperpolarization.

A Tetrahymena Piwi bound to mature tRNA 3' fragments activates the exonuclease Xrn2 for RNA processing in the nucleus.

Emerging evidence suggests that Argonaute (Ago)/Piwi proteins have diverse functions in the nucleus and cytoplasm, but the molecular mechanisms employed in the nucleus remain poorly defined. The Tetrahymena thermophila Ago/Piwi protein Twi12 is essential for growth and functions in the nucleus. Twi12-bound small RNAs (sRNAs) are 3' tRNA fragments that contain modified bases and thus are attenuated for base pairing to targets. We show that Twi12 assembles an unexpected complex with the nuclear exonuclease Xrn2. Twi12 functions to stabilize and localize Xrn2, as well as to stimulate its exonuclease activity. Twi12 function depends on sRNA binding, which is required for its nuclear import. Depletion of Twi12 or Xrn2 induces a cellular ribosomal RNA processing defect known to result from limiting Xrn2 activity in other organisms. Our findings suggest a role for an Ago/Piwi protein and 3' tRNA fragments in nuclear RNA metabolism.

Effects of the aquatic contaminant human pharmaceuticals and their mixtures on the proliferation and migratory responses of the bioindicator freshwater ciliate Tetrahymena.

An increasing attention is paid to the potential harmful effects of aquatic contaminant pharmaceuticals exerted on both biosystems and humans. In the present work the effects of 14 pharmaceuticals including NSAIDs, antibiotics, ß-blockers and a frequently used X-ray contrast media on the proliferation and migratory behavior of the freshwater ciliate Tetrahymena pyriformis was investigated. Moreover, the mixture toxicity of four selected pharmaceuticals (diclofenac, ibuprofen, metoprolol and propranolol) was evaluated in binary mixtures using full factorial experimental design. Our results showed that the sensitivity of Tetrahymena to NSAIDs and ß-blockers (EC(50) ranged from 4.8 mg L(-1) to 308.1 mg L(-1)) was comparable to that of algal or Daphnia bioassays. Based on these elevated EC(50) values acute toxic effects of these pharmaceuticals to T. pyriformis are unlikely. Antibiotics and the contrast agent sodium-diatrizoate had no proliferation inhibiting effect. Chemotactic response of Tetrahymena was more sensible than proliferation as significant chemorepellent action was observed in the environmentally realistic concentration range for acetylsalicylic acid, diclofenac, fenoprofen, paracetamol, metoprolol, propranolol, timolol and trimethoprim (Chemotaxis Index ranged from 63% to 88%). Mixture toxicity experiments resulted in a complex, concentration dependent interaction type pattern with antagonism being the predominant interaction type (59%) followed by additivity (37%) and synergism (4%). Hence the concept of concentration addition validated for NSAIDs in other organisms cannot be adopted for this ciliate. In summary authors suggest Tetrahymena as a sensible model of testing aquatic contaminants as well as underline the significance using more specific endpoints to understand the complex mechanisms investigated.

A simple microscopy assay to teach the processes of phagocytosis and exocytosis.

Phagocytosis and exocytosis are two cellular processes involving membrane dynamics. While it is easy to understand the purpose of these processes, it can be extremely difficult for students to comprehend the actual mechanisms. As membrane dynamics play a significant role in many cellular processes ranging from cell signaling to cell division to organelle renewal and maintenance, we felt that we needed to do a better job of teaching these types of processes. Thus, we developed a classroom-based protocol to simultaneously study phagocytosis and exocytosis in Tetrahymena pyriformis. In this paper, we present our results demonstrating that our undergraduate classroom experiment delivers results comparable with those acquired in a professional research laboratory. In addition, students performing the experiment do learn the mechanisms of phagocytosis and exocytosis. Finally, we demonstrate a mathematical exercise to help the students apply their data to the cell. Ultimately, this assay sets the stage for future inquiry-based experiments, in which the students develop their own experimental questions and delve deeper into the mechanisms of phagocytosis and exocytosis.

Predation influences the structure of biofilm developed on ultrafiltration membranes.

This study investigates the impact of predation by eukaryotes on the development of specific biofilm structures in gravity-driven dead-end ultrafiltration systems. Filtration systems were operated under ultra-low pressure conditions (65 mbar) without the control of biofilm formation. Three different levels of predation were evaluated: (1) inhibition of eukaryotic organisms, (2) addition of cultured protozoa (Tetrahymena pyriformis), and (3) no modification of microbial community as a control. The system performance was evaluated based on permeate flux and structures of the biofilm. It was found that predation had a significant influence on both the total amount and also the structure of the biofilm. An open and heterogeneous structure developed in systems with predation whereas a flat, compact, and thick structure that homogeneously covered the membrane surface developed in absence of predation. Permeate flux was correlated with the structure of the biofilm with increased fluxes for smaller membrane coverage. Permeate fluxes in the presence or absence of the predators was 10 and 5 L m(-2) h(-1), respectively. It was concluded that eukaryotic predation is a key factor influencing the performance of gravity-driven ultrafiltration systems.

A knockout mutation of a constitutive GPCR in Tetrahymena decreases both G-protein activity and chemoattraction.

Although G-protein coupled receptors (GPCRs) are a common element in many chemosensory transduction pathways in eukaryotic cells, no GPCR or regulated G-protein activity has yet been shown in any ciliate. To study the possible role for a GPCR in the chemoresponses of the ciliate Tetrahymena, we have generated a number of macronuclear gene knockouts of putative GPCRs found in the Tetrahymena Genome database. One of these knockout mutants, called G6, is a complete knockout of a gene that we call GPCR6 (TTHERM_00925490). Based on sequence comparisons, the Gpcr6p protein belongs to the Rhodopsin Family of GPCRs. Notably, Gpcr6p shares highest amino acid sequence homologies to GPCRs from Paramecium and several plants. One of the phenotypes of the G6 mutant is a decreased responsiveness to the depolarizing ions Ba²âº and K⁺, suggesting a decrease in basal excitability (decrease in Ca²âº channel activity). The other major phenotype of G6 is a loss of chemoattraction to lysophosphatidic acid (LPA) and proteose peptone (PP), two known chemoattractants in Tetrahymena. Using microsomal [³5S]GTPγS binding assays, we found that wild-type (CU427) have a prominent basal G-protein activity. This activity is decreased to the same level by pertussis toxin (a G-protein inhibitor), addition of chemoattractants, or the G6 mutant. Since the basal G-protein activity is decreased by the GPCR6 knockout, it is likely that this gene codes for a constitutively active GPCR in Tetrahymena. We propose that chemoattractants like LPA and PP cause attraction in Tetrahymena by decreasing the basal G-protein stimulating activity of Gpcr6p. This leads to decreased excitability in wild-type and longer runs of smooth forward swimming (less interrupted by direction changes) towards the attractant. Therefore, these attractants may work as inverse agonists through the constitutively active Gpcr6p coupled to a pertussis-sensitive G-protein.