The Tetrahymena argonaute-binding protein Giw1p directs a mature argonaute-siRNA complex to the nucleus.

Emerging evidence suggests that RNA interference (RNAi)-related processes act both in the cytoplasm and in the nucleus. However, the process by which the RNAi machinery is transported into the nucleus remains poorly understood. The Tetrahymena Argonaute protein Twi1p localizes to the nucleus and is crucial for small RNA-directed programmed DNA elimination. In this study, we identify Giw1p, which binds to Twi1p and is required for its nuclear localization. Furthermore, the endoribonuclease (Slicer) activity of Twi1p plays a vital role in the removal of one of the two strands of Twi1p-associated small interfering RNAs (siRNAs), leading to a functionally mature Twi1p-siRNA complex. Slicer activity is also shown to be required for nuclear localization of Twi1p and for its association with Giw1p. These results suggest that Giw1p senses the state of Twi1p-associated siRNAs and selectively transports the mature Twi1p-siRNA complex into the nucleus.

Boundaries of eliminated heterochromatin of Tetrahymena are positioned by the DNA-binding protein Ltl1.

During differentiation of the Tetrahymena thermophila somatic nucleus, its germline-derived DNA undergoes extensive reorganization including the removal of ∼50 Mb from thousands of loci called internal eliminated sequences (IESs). IES-associated chromatin is methylated on lysines 9 and 27 of histone H3, marking newly formed heterochromatin for elimination. To ensure that this reorganized genome maintains essential coding and regulatory sequences, the boundaries of IESs must be accurately defined. In this study, we show that the developmentally expressed protein encoded by Lia3-Like 1 (LTL1) (Ttherm_00499370) is necessary to direct the excision boundaries of particular IESs. In ΔLTL1 cells, boundaries of eliminated loci are aberrant and heterogeneous. The IESs regulated by Ltl1 are distinct from those regulated by the guanine-quadruplex binding Lia3 protein. Ltl1 has a general affinity for double stranded DNA (Kd ∼ 350 nM) and binds specifically to a 50 bp A+T rich sequence flanking each side of the D IES (Kd ∼ 43 nM). Together these data reveal that Ltl1 and Lia3 control different subsets of IESs and that their mechanisms for flanking sequence recognition are distinct.

The nonhistone, N-terminal tail of an essential, chimeric H2A variant regulates mitotic H3-S10 dephosphorylation.

H2A.Y is an essential, divergent Tetrahymena thermophila histone variant. It has a long nonhistone N terminus that contains leucine-rich repeats (LRR) and an LRR cap domain with similarity to Sds22p, a regulator of yeast protein phosphatase 1 (PP1) activity in the nucleus. In growing cells, H2A.Y is incorporated into micronuclei only during S phase, which occurs immediately after micronuclear mitosis. Depletion of H2A.Y causes prolonged retention of mitosis-associated histone H3-S10 phosphorylation and mitotic abnormalities that mimic S10E mutation. In cells where H2A.Y is depleted, an inducible chimeric gene, in which the H2A.Y N terminus is attached to H2A.X, is shown to regulate micronuclear H3-S10 phosphorylation. H2A.Y can also be specifically coimmunoprecipitated with a Tetrahymena PP1 ortholog (Ppo1p). Taken together, these results argue that the N terminus of H2A.Y functions to regulate H3-S10 dephosphorylation. This striking in vivo case of "cross-talk" between a H2A variant and a specific post-translational modification of another histone demonstrates a novel function for a histone variant.

[Inducible Expression of Ran1 and Its GDP- and GTP-Bound Mimetic Mutants Leads to Defects in Amitosis and Cytokinesis with Abnormal Cytoplasmic Microtubule Assembly].

Ran is an evolutionarily conserved GTPase crucial in regulating various cell divisions, including mitosis and meiosis. A previous study showed that the knockdown of RAN1 inhibited macronuclear amitosis with the abnormal organization of intramacronuclear microtubules in Tetrahymena thermophila. This study aimed to further investigate the effects of the inducible expression of wild-type Ran1 (Ran1WT), GTP-bound Ran1-mimetic (Ran1Q70L), and GDP-bound Ran1-mimetic (Ran1T25N) on cytoplasmic microtubule assembly during amitosis of T. thermophila, based on previous studies about their effects on intramacronuclear microtubule. The mutant strains of T. thermophila for inducible expression of Ran1WT/T25N/Q70L by Cd^(2+) were constructed. The inducibly expressed HA-Ran1Q70L/T25N distributed asymmetrically across the macronuclear envelope during amitosis. At the lower level of inducible expression, only Ran1T25N showed a significant decreasing effect on T. thermophila reproduction, macronuclear amitosis and cytokinesis. At the higher level of inducible expression, Ran1WT/Q70L/T25N inhibited T. thermophila reproduction, macronuclear amitosis and cytokinesis, and the inhibitive effect of Ran1T25N was the most significant. The inducible expression of Ran1WT/Q70L/T25N led to defects in amitosis and cytokinesis with abnormal cytoplasmic microtubule assembly. These results further confirmed the regulatory function of Ran1 on amitosis and suggested a novel role of Ran1 in cytokinesis and the alignment of cytoplasmic microtubules in T. thermophila.

Coiled-coil domain containing 42 (Ccdc42) is necessary for proper sperm development and male fertility in the mouse.

Spermiogenesis is the differentiation of spermatids into motile sperm consisting of a head and a tail. The head harbors a condensed elongated nucleus partially covered by the acrosome-acroplaxome complex. Defects in the acrosome-acroplaxome complex are associated with abnormalities in sperm head shaping. The head-tail coupling apparatus (HTCA), a complex structure consisting of two cylindrical microtubule-based centrioles and associated components, connects the tail or flagellum to the sperm head. Defects in the development of the HTCA cause sperm decapitation and disrupt sperm motility, two major contributors to male infertility. Here, we provide data indicating that mutations in the gene Coiled-coil domain containing 42 (Ccdc42) is associated with malformation of the mouse sperm flagella. In contrast to many other flagella and motile cilia genes, Ccdc42 expression is only observed in the brain and developing sperm. Male mice homozygous for a loss-of-function Ccdc42 allele (Ccdc42(KO)) display defects in the number and location of the HTCA, lack flagellated sperm, and are sterile. The testes enriched expression of Ccdc42 and lack of other phenotypes in mutant mice make it an ideal candidate for screening cases of azoospermia in humans.

Kinesin-14 is Important for Chromosome Segregation During Mitosis and Meiosis in the Ciliate Tetrahymena thermophila.

Ciliates such as Tetrahymena thermophila have two distinct nuclei within one cell: the micronucleus that undergoes mitosis and meiosis and the macronucleus that undergoes amitosis, a type of nuclear division that does not involve a bipolar spindle, but still relies on intranuclear microtubules. Ciliates provide an opportunity for the discovery of factors that specifically contribute to chromosome segregation based on a bipolar spindle, by identification of factors that affect the micronuclear but not the macronuclear division. Kinesin-14 is a conserved minus-end directed microtubule motor that cross-links microtubules and contributes to the bipolar spindle sizing and organization. Here, we use homologous DNA recombination to knock out genes that encode kinesin-14 orthologues (KIN141, KIN142) in Tetrahymena. A loss of KIN141 led to severe defects in the chromosome segregation during both mitosis and meiosis but did not affect amitosis. A loss of KIN141 altered the shape of the meiotic spindle in a way consistent with the KIN141's contribution to the organization of the spindle poles. EGFP-tagged KIN141 preferentially accumulated at the spindle poles during the meiotic prophase and metaphase I. Thus, in ciliates, kinesin-14 is important for nuclear divisions that involve a bipolar spindle.

Mutational robustness of morphological traits in the ciliate Tetrahymena thermophila.

Ciliate nuclear architecture, in particular the sequestration of a transcriptionally silent germline genome, allows for the accumulation of mutations that are "hidden" from selection during many rounds of asexual reproduction. After sexual conjugation, these mutations are expressed, potentially resulting in highly variable phenotypes. Morphological traits are widely used in ciliate taxonomy, however, the extent to which the values of these traits are robust to change in the face of mutation remains largely unknown. In this study, we examine the effects of mutations accumulated in the germline genome to test the mutational robustness of four traits commonly used in ciliate morphological taxonomy (number of somatic kineties, number of postoral kineties, macronuclear size, and cell size). We found that the number of postoral kineties is robust to mutation, confirming that it should be preferentially used in taxonomy. By contrast, we found that, as in other unicellular and multicellular species, cell and macronucleus sizes change in response to mutation. Thus, we argue that cell and macronucleus sizes, which are widely used in taxonomy, should be treated cautiously for species identification. Finally, we found evidence of correlations between cell and macronucleus sizes and fitness, suggesting possible mutational pleiotropy. This study demonstrates the importance of, and methods for, determining mutational robustness to guide morphological taxonomy in ciliates.

A novel mitochondrial nuclease-associated protein: a major executor of the programmed nuclear death in Tetrahymena thermophila.

BACKGROUND INFORMATION: Programmed nuclear death (PND) in the ciliate Tetrahymena is an apoptosis-like phenomenon that occurs in a restricted space of cytoplasm during conjugation. In the process, only the parental macronucleus is selectively eliminated from the progeny cytoplasm, in conjunction with differentiation of new macronuclei for the next generation. For the last decade, mitochondria have been elucidated to be a crucial executioner like apoptosis: apoptosis-inducing factor and yet-unidentified nucleases localised in mitochondria are major factors for PND. RESULTS: To identify such nucleases, we performed a DNase assay in a PAGE (SDS-DNA-PAGE) using total mitochondrial proteins. Some proteins showed DNase activity, but particularly a 17 kDa protein exhibited the highest and predominant activity. Mass spectrometric analysis revealed a novel mitochondrial nuclease, named TMN1, whose homologue has been discovered only in the ciliate Paramecium tetraurelia, but not in other eukaryotes. Gene disruption of TMN1 led to a drastic reduction of mitochondrial nuclease activity and blocked nuclear degradation during conjugation, but did not affect accumulation of autophagic and lysosomal machinery around the parental macronucleus. CONCLUSIONS: These observations strongly suggest that the mitochondrial nuclease-associated protein plays a key role in PND as a major executor. Taking the novel protein specific to ciliates in consideration, Tetrahymena would have diverted a different protein from common apoptotic factors shared in eukaryotes to PND in the course of ciliate evolution.

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.

TiO2 nanoparticles act as a carrier of Cd bioaccumulation in the ciliate Tetrahymena thermophila.

When nanoparticles can enter a unicellular organism directly, how may they affect the bioaccumulation and toxicity of other pollutants already present in the environment? To answer this question, we conducted experiments with a protozoan Tetrahymena thermophila. The well-dispersed polyacrylate-coated TiO2 nanoparticles (PAA-TiO2-NPs) were used as a representative nanomaterial, and Cd as a conventional pollutant. We found that PAA-TiO2-NPs could get into Tetrahymena cells directly. Such internalization was first induced by low concentrations of Cd, but later suppressed when Cd concentrations were higher than 1 µg/L. Considering its significant adsorption on PAA-TiO2-NPs, Cd could be taken up by T. thermophila in the form of free ion or metal-nanoparticle complexes. The latter route accounted for 46.3% of Cd internalization. During the 5 h depuration period, 4.34-22.1% of Cd was excreted out, which was independent of the concentrations of intracellular Cd and PAA-TiO2-NPs. On the other hand, both free and intracellular Cd concentrations only partly predicted its toxicity at different levels of PAA-TiO2-NPs. This may have resulted from PAA-TiO2-NPs' synergistic effects and the distinct subcellular distribution of Cd taken up via the two routes above. Overall, we should pay attention to the carrier effects of nanoparticles when assessing their environmental risks.

Functional GFP-metallothionein fusion protein from Tetrahymena thermophila: a potential whole-cell biosensor for monitoring heavy metal pollution and a cell model to study metallothionein overproduction effects.

The significance of metal(oid)s as environmental pollutants has made them a priority in ecotoxicology, with the aim of minimizing exposure to animals or humans. Therefore, it is necessary to develop sensitive and inexpensive methods that can efficiently detect and monitor these pollutants in the environment. Conventional analytical techniques suffer from the disadvantages of high cost and complexity. Alternatively, prokaryotic or eukaryotic whole-cell biosensors (WCB) are one of the newest molecular tools employed in environmental monitoring that use the cell as an integrated reporter incorporating a reporter gene fused to a heavy metal responsive promoter. In the present paper, we report results from expressing, in the ciliate Tetrahymena thermophila, constructs consisting of the reporter gfp gene fused to the complete MTT1 or MTT5 protein coding regions under the transcriptional control of the MTT1 metallothionein promoter, which plays a critical role in heavy metal stress in this ciliate. When exposed to Cd(2+), such cells overexpress both the GFP reporter transgene and the linked metallothionein gene. We report that, for the GFPMTT5 strain, this metallothionein overexpression results in marked resistance to cadmium toxicity (24 h LC50 ~15 µM of Cd(2+)), compared to wild type cells (24 h LC50 ~1.73 µM of Cd(2+)). These results provide the first experimental evidence that ciliate metallothioneins, like in other organisms, function to protect the cell against toxic metal ions. Because these strains may have novel advantages as WCBs, we have compared their properties to those of other previously reported Tetrahymena WCBs.

A microfluidic-enabled mechanical microcompressor for the immobilization of live single- and multi-cellular specimens.

A microcompressor is a precision mechanical device that flattens and immobilizes living cells and small organisms for optical microscopy, allowing enhanced visualization of sub-cellular structures and organelles. We have developed an easily fabricated device, which can be equipped with microfluidics, permitting the addition of media or chemicals during observation. This device can be used on both upright and inverted microscopes. The apparatus permits micrometer precision flattening for nondestructive immobilization of specimens as small as a bacterium, while also accommodating larger specimens, such as Caenorhabditis elegans, for long-term observations. The compressor mount is removable and allows easy specimen addition and recovery for later observation. Several customized specimen beds can be incorporated into the base. To demonstrate the capabilities of the device, we have imaged numerous cellular events in several protozoan species, in yeast cells, and in Drosophila melanogaster embryos. We have been able to document previously unreported events, and also perform photobleaching experiments, in conjugating Tetrahymena thermophila.

PHLP2 is essential and plays a role in ciliogenesis and microtubule assembly in Tetrahymena thermophila.

Recent studies have implicated the phosducin-like protein-2 (PHLP2) in regulation of CCT, a chaperonin whose activity is essential for folding of tubulin and actin. However, the exact molecular function of PHLP2 is unclear. Here we investigate the significance of PHLP2 in a ciliated unicellular model, Tetrahymena thermophila, by deleting its single homolog, Phlp2p. Cells lacking Phlp2p became larger and died within 96 h. Overexpressed Phlp2p-HA localized to cilia, basal bodies, and cytosol without an obvious change in the phenotype. Despite similar localization, overexpressed GFP-Phlp2p caused a dominant-negative effect. Cells overproducing GFP-Phlp2p had decreased rates of proliferation, motility and phagocytosis, as compared to wild type cells or cells overproducing a non-tagged Phlp2p. Growing GFP-Phlp2p-overexpressing cells had fewer cilia and, when deciliated, failed to regenerate cilia, indicating defects in cilia assembly. Paclitaxel-treated GFP-Phlp2p cells failed to elongate cilia, indicating a change in the microtubules dynamics. The pattern of ciliary and cytosolic tubulin isoforms on 2D gels differed between wild type and GFP-Phlp2p-overexpressing cells. Thus, in Tetrahymena, PhLP2 is essential and under specific experimental conditions its activity affects tubulin and microtubule-dependent functions including cilia assembly.

Differential targeting of Tetrahymena ORC to ribosomal DNA and non-rDNA replication origins.

The Tetrahymena thermophila origin recognition complex (ORC) contains an integral RNA subunit, 26T RNA, which confers specificity to the amplified ribosomal DNA (rDNA) origin by base pairing with an essential cis-acting replication determinant--the type I element. Using a plasmid maintenance assay, we identified a 6.7 kb non-rDNA fragment containing two closely associated replicators, ARS1-A (0.8 kb) and ARS1-B (1.2 kb). Both replicators lack type I elements and hence complementarity to 26T RNA, suggesting that ORC is recruited to these sites by an RNA-independent mechanism. Consistent with this prediction, although ORC associated exclusively with origin sequences in the 21 kb rDNA minichromosome, the interaction between ORC and the non-rDNA ARS1 chromosome changed across the cell cycle. In G(2) phase, ORC bound to all tested sequences in a 60 kb interval spanning ARS1-A/B. Remarkably, ORC and Mcm6 associated with just the ARS1-A replicator in G(1) phase when pre-replicative complexes assemble. We propose that ORC is stochastically deposited onto newly replicated non-rDNA chromosomes and subsequently targeted to preferred initiation sites prior to the next S phase.

Differential growth of and nanoscale TiO2 accumulation in Tetrahymena thermophila by direct feeding versus trophic transfer from Pseudomonas aeruginosa.

Nanoscale titanium dioxide (TiO2) is increasingly used in consumer goods and is entering waste streams, thereby exposing and potentially affecting environmental microbes. Protozoans could either take up TiO2 directly from water and sediments or acquire TiO2 during bactivory (ingestion of bacteria) of TiO2-encrusted bacteria. Here, the route of exposure of the ciliated protozoan Tetrahymena thermophila to TiO2 was varied and the growth of, and uptake and accumulation of TiO2 by, T. thermophila were measured. While TiO2 did not affect T. thermophila swimming or cellular morphology, direct TiO2 exposure in rich growth medium resulted in a lower population yield. When TiO2 exposure was by bactivory of Pseudomonas aeruginosa, the T. thermophila population yield and growth rate were lower than those that occurred during the bactivory of non-TiO2-encrusted bacteria. Regardless of the feeding mode, T. thermophila cells internalized TiO2 into their food vacuoles. Biomagnification of TiO2 was not observed; this was attributed to the observation that TiO2 appeared to be unable to cross the food vacuole membrane and enter the cytoplasm. Nevertheless, our findings imply that TiO2 could be transferred into higher trophic levels within food webs and that the food web could be affected by the decreased growth rate and yield of organisms near the base of the web.

Effects of a new photoactivatable cationic porphyrin on ciliated protozoa and branchiopod crustaceans, potential components of freshwater ecosystems polluted by pathogenic agents and their vectors.

The increasing use of photosensitized processes for disinfection of microbiologically polluted waters requires a precise definition of the factors controlling the degree of photosensitivity in target and non-target organisms. In this regard, tests with protozoa and invertebrates which have a natural habitat in such waters may be used as first screening methods for the assessment of possible hazards for the ecosystem. A new cationic porphyrin, namely meso-tri(N-methyl-pyridyl)mono(N-dodecyl-pyridyl)porphine (C12), is tested in this work on the protozoan Ciliophora Colpoda inflata and Tetrahymena thermophila and the Crustacea Branchiopoda Artemia franciscana and Daphnia magna. The protocol involved 1 h incubation with porphyrin doses in the 0.1-10.0 µM range and subsequent irradiation with visible light at a fluence rate of 10 mW cm(-2). The results indicate that C12 porphyrin has a significant affinity for C. inflata and T. thermophila; this is also shown by fluorescence microscopic analyses. C. inflata cysts were resistant to the phototreatment up to a porphyrin dose of 0.6 µM. The effects of C12 on cysts have been evaluated at 3 and 24 h after the end of the phototreatment; a delay in the excystment process was observed. T. thermophila was fairly resistant to the phototreatment with C12 porphyrin. The data obtained with the two crustaceans indicated that the effects of dark- and photo-treatment with C12 need to be closely examined for every organism. A. franciscana is more resistant, probably owing to its ability to adapt to extreme conditions, while the high level of photosensitivity displayed by Daphnia magna represents a potential drawback, as this organism is often selected as a reference standard for assessing the environmental safety. Thus, while C12 photosensitisation can represent a useful tool for inducing a microbicidal or larvicidal action on polluted waters, the irradiation protocols must be carefully tailored to the nature of the specific water basin, and in particular to its biotic characteristics.

A dynamin-related protein required for nuclear remodeling in Tetrahymena.

Dynamin-related proteins (DRPs) are GTPases that reversibly assemble on cellular membranes [1]. Individual DRPs (here "DRP" includes authentic dynamins) function in fission or tubulation of the plasma membrane, trans-Golgi network, mitochondria, peroxisomes, chloroplasts, and endosomes [1] and in mitochondrial fusion [2]. Many of these functions are widespread; they are present in animals, plants, trypanosomes, Giardia, ciliates, alga, and slime molds [3-8]. Lineage-specific expansions of the gene family created specialized DRPs. In animals, such DRPs include MxB, which has been reported to regulate nuclear-pore transport [9]. Whereas many unicellular organisms possess a small number of DRPs, expansions occurred in some protist lineages. The eight DRPs in the ciliate Tetrahymena thermophila might contribute to aspects of ciliate complexity. Each ciliate cell contains distinct germline and somatic nuclei, whose differentiation and maintenance must require distinct machinery [10, 11]. Here we show that Drp6p, previously shown to be targeted to the nuclear envelope [3], is required for macronuclear development. Drp6p activity, which is distinct from that of the only other known nuclear DRP, is modulated by a combination of stage-specific subcellular targeting and assembly dynamics. This work demonstrates a novel DRP activity and presents a system in which environmental and developmental cues can be used for manipulating key aspects of regulation.

Ciliate biology: dynamin goes nuclear.

Dynamin and dynamin-related proteins (DRPs) mediate an array of membrane fission processes. A Tetrahymena DRP has adopted a new role, assisting in nuclear differentiation, a finding that further highlights these proteins - and this ciliate - as biological innovators.

Single-cell isolation and cloning of Tetrahymena thermophila cells with a fluorescence-activated cell sorter.

We developed a method for cloning cells of the ciliate Tetrahymena thermophila in chemically defined medium (CDM) using a fluorescence-activated cell sorter (FACS). Although T. thermophila is a model unicellular eukaryote, two major technical difficulties remain in its cloning. First, T. thermophila fails to proliferate from low density in CDM, particularly if the inoculum contains single cells. Second, general cloning methods are time consuming and have low throughput. Here, we modified the CDM by addition of bovine serum albumin that helped growth from an inoculum with a density of 10 cell/ml (1 cell/100 µl). In addition, we applied a FACS for isolation of single cells. We showed that it is possible to separate cell populations based on the presence or absence of phagocytosed fluorescent beads and to isolate single cells in a modified CDM by FACS. Our techniques allow the direct isolation of single cells and facilitate the establishment of clonal strains.

A developmentally regulated gene, ASI2, is required for endocycling in the macronuclear anlagen of Tetrahymena.

Ciliated protozoa contain two types of nuclei, germ line micronuclei (Mic) and transcriptionally active macronuclei (Mac). During sexual reproduction, the parental Mac degenerates and a new Mac develops from a mitotic product of the zygotic Mic. Macronuclear development involves extensive endoreplication of the genome. The present study shows that endoreplication of macronuclear DNA in Tetrahymena is an example of endocyling, a variant of the mitotic cycle with alternating S and G phases in the absence of cell division. Thus, endocycling is conserved from ciliates to multicellular organisms. The gene ASI2 in Tetrahymena thermophila encodes a putative signal transduction receptor. ASI2 is nonessential for vegetative growth, but it is upregulated during development of the new Mac. Cells that lack ASI2 in the developing Mac anlagen are arrested in endoreplication of the DNA and die. This study shows that ASI2 is also transcribed in the parental Mac early in conjugation and that transcription of ASI2 in the parental Mac supports endoreplication of the DNA during early stages of development of the Mac anlagen. Other molecular events in Mac anlage development, including developmentally regulated DNA rearrangement, occur normally in matings between ASI2 knockouts, suggesting that ASI2 specifically regulates endocycling in Tetrahymena.