Kensuke Kataoka

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.

Small-RNA-Mediated Genome-wide trans-Recognition Network in Tetrahymena DNA Elimination

Summary Small RNAs are used to silence transposable elements (TEs) in many eukaryotes, which use diverse evolutionary solutions to identify TEs. In ciliated protozoans, small-RNA-mediated comparison of the germline and somatic genomes underlies identification of TE-related sequences, which are then eliminated from the soma. Here, we describe an additional mechanism of small-RNA-mediated identification of TE-related sequences in the ciliate Tetrahymena. We show that a limited set of internal eliminated sequences (IESs) containing potentially active TEs produces a class of small RNAs that recognize not only the IESs from which they are derived, but also other IESs in trans. This trans recognition triggers the expression of yet another class of small RNAs that identify other IESs. Therefore, TE-related sequences in Tetrahymena are robustly targeted for elimination by a genome-wide trans-recognition network accompanied by a chain reaction of small RNA production.

Presence of aquaporin and V-ATPase on the contractile vacuole of Amoeba proteus.

Background information. The results of water permeability measurements suggest the presence of an AQP (aquaporin) in the membrane of the CV (contractile vacuole) in Amoeba proteus [Nishihara, Shimmen and Sonobe ( 2004 ) Cell Struct. Funct. 29, 85–90].

Modules for C-terminal epitope tagging of Tetrahymena genes

Although epitope tagging has been widely used for analyzing protein function in many organisms, there are few genetic tools for epitope tagging in Tetrahymena. In this study, we describe several C-terminal epitope tagging modules that can be used to express tagged proteins in Tetrahymena cells by both plasmid- and PCR-based strategies.

Phosphorylation of an HP1-like Protein Regulates Heterochromatin Body Assembly for DNA Elimination

Summary Heterochromatic loci are often assembled into higher-order heterochromatin bodies in diverse eukaryotes. However, the formation and biological roles of heterochromatin bodies are poorly understood. In the ciliated protozoan Tetrahymena, de novo heterochromatin body formation is accompanied by programmed DNA elimination. Here, we show that the heterochromatin body component Jub1p promotes heterochromatin body formation and dephosphorylation of the Heterochromatin Protein 1-like protein Pdd1p. Through the mutagenesis of the phosphorylated residues of Pdd1p, we demonstrate that Pdd1p dephosphorylation promotes the electrostatic interaction between Pdd1p and RNA in vitro and heterochromatin body formation in vivo. We therefore propose that heterochromatin body is assembled by the Pdd1p-RNA interaction. Pdd1p dephosphorylation and Jub1p are required for heterochromatin body formation and DNA elimination but not for local heterochromatin assembly, indicating that heterochromatin body plays an essential role in DNA elimination.

Developmental regulation of locomotive activity in Xenopus primordial germ cells

Primordial germ cells (PGCs) arise in the early embryo and migrate toward the future gonad through species‐specific pathways. They are assumed to change their migration properties dependent on their own genetic program and/or environmental cues, though information concerning the developmental change in PGC motility is limited. First, we re‐examined the distribution of PGCs in the endodermal region of Xenopus embryos at various stages by using an antibody against Xenopus Daz‐like protein, and found four stages of migration, namely clustering, dispersing, directionally migrating and re‐aggregating. Next, we isolated living PGCs at each stage and directly examined their morphology and locomotive activity in cell cultures. PGCs at the clustering stage were round in shape with small blebs and showed little motility. PGCs in both the dispersing and the directionally migrating stages alternated between the locomotive phase with an elongated morphology and the pausing phase with a rugged morphology. The locomotive activity of the elongated PGCs was accompanied by the persistent formation of a large bleb at the leading front. The duration of the locomotive phase was shortened gradually with the transition from the dispersing stage to the directionally migrating stage. At the re‐aggregating stage, PGCs became round in shape and showed no motility. Thus, we directly showed that the locomotive activity of PGCs changes dynamically depending upon the migrating stage. We also showed that the locomotion and blebbing of the PGCs required F‐actin, myosin II activity and RhoA/Rho‐associated protein kinase (ROCK) signaling.

Perturbation of Notch/Suppressor of Hairless pathway disturbs migration of primordial germ cells in Xenopus embryo.

Primordial germ cells (PGCs) in Xenopus embryo are specified in the endodermal cell mass and migrate dorsally toward the future gonads. The role of the signal mediated by Notch and Suppressor of Hairless [Su(H)] was analyzed on the migrating PGCs at the tailbud stage. X‐Notch‐1 and X‐Delta‐1 are expressed in the migrating PGCs and surrounding endodermal cells, whereas X‐Delta‐2 and X‐Serrate‐1 are expressed preferentially in the PGCs. Suppression and constitutive activation of the Notch/Su(H) signaling in the whole endoderm region or selectively in the PGCs resulted in an increase in ectopic PGCs located in lateral or ventral regions. Knocking down of the Notch ligands by morpholino oligonucleotides revealed that X‐Delta‐2 was indispensable for the correct PGC migration. The ectopic PGCs seemed to have lost their motility in the Notch/Su(H) signal‐manipulated embryos. Our results suggest that a cell‐to‐cell interaction via the Notch/Su(H) pathway has a significant role in the PGC migration by regulating cell motility.

Identification of asymmetrically localized transcripts along the animal–vegetal axis of the Xenopus egg

In many organisms, proper embryo development depends on the asymmetrical distribution of mRNA in the cytoplasm of the egg. Here we report comprehensive screening of RNA localized in the animal or vegetal hemisphere of the Xenopus egg. Macroarrays including over 40 000 independent embryonic cDNA clones, representing at least 17 000 unigenes, were differentially hybridized with labeled probes synthesized from the mRNA of animal or vegetal blastomeres. After two rounds of screening, we identified 33 clones of transcripts that may be preferentially distributed in the vegetal region of the early stage embryo, but transcripts localized in the animal region were not found. To assess the array results, we performed northern blot and quantitative real‐time reverse transcription–polymerase chain reaction analysis. As a result, 21 transcripts of the 33 were confirmed to be localized in the vegetal region of the early stage embryo. Whole‐mount in situ hybridization analysis revealed that 11 transcripts, including 7 previously reported genes, were localized in the vegetal hemisphere of the egg. These 11 transcripts were categorized into three groups according to their expression patterns in the egg. The first group, which contained four transcripts, showed uniform expression in the vegetal hemisphere, similar to VegT. The second group, which contained three transcripts, showed gradual expression from the vegetal pole to the equator, similar to Vg1. The last group, which contained three transcripts, was expressed at the germ plasm, similar to Xdazl. One transcript, Xwnt11, showed both the second and the third expression patterns.

A Zip3-like protein plays a role in crossover formation in the SC-less meiosis of the protist Tetrahymena

Tetrahymena has a crossover pathway that combines features of both canonical pathways found in the majority of eukaryotes. The pathway uses a protein related to Zip3, which is commonly associated with the formation of a synaptonemal complex.

Negative Regulators of an RNAi-Heterochromatin Positive Feedback Loop Safeguard Somatic Genome Integrity in Tetrahymena

Summary RNAi-mediated positive feedback loops are pivotal for the maintenance of heterochromatin, but how they are downregulated at heterochromatin-euchromatin borders is not well understood. In the ciliated protozoan Tetrahymena, heterochromatin is formed exclusively on the sequences that are removed from the somatic genome by programmed DNA elimination, and an RNAi-mediated feedback loop is important for assembling heterochromatin on the eliminated sequences. In this study, we show that the heterochromatin protein 1 (HP1)-like protein Coi6p, its interaction partners Coi7p and Lia5p, and the histone demethylase Jmj1p are crucial for confining the production of small RNAs and the formation of heterochromatin to the eliminated sequences. The loss of Coi6p, Coi7p, or Jmj1p causes ectopic DNA elimination. The results provide direct evidence for the existence of a dedicated mechanism that counteracts a positive feedback loop between RNAi and heterochromatin at heterochromatin-euchromatin borders to maintain the integrity of the somatic genome.