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Kanzaki, Natsumi, Hoshi, Yuki, Kumagai, Ryohei, Sugimoto, Asako, Kikuchi, Taisei, Namai, Satoshi, Tsuyama, Kenji
[
International Worm Meeting,
2017]
Caenorhabditis sp. 34 is a sister species of C. elegans recently isolated from the syconia of the fig Ficus septica on Ishigaki Island, Japan (see abstract by T. Kikuchi, et al.). C. sp. 34 is gonochoric and shares typological key characters with other Elegans supergroup species, but strikingly, adults are nearly twice as long as C. elegans. The optimal culture temperature for C. sp. 34 is significantly higher (27 deg C) than that of C. elegans (20 deg C). Young adult males and females tend to form clumps, and Dauer larvae are rarely observed in laboratory culture conditions. Recently the C. sp. 34 genome assembly was produced into six chromosomes (see abstract by T. Kikuchi, et al.). The marked differences from C. elegans in morphology, behaviors and ecology, and the availability of the complete genome sequence make C. sp. 34 highly attractive for comparative and evolutionary studies. To make C. sp. 34 genetically tractable, we have been developing genetic and molecular techniques and tools. Stable transgenic lines of C. sp.34 could be obtained by microinjecting marker plasmids commonly used in C. elegans, although the efficiency was lower than that in C. elegans. Both soaking and feeding RNAi was as effective as in C. elegans. A panel of antibodies against C. elegans proteins successfully recognized expected structures in C. sp. 34 by immunofluorescence. Thus, many of the rich genetic and molecular resources for C. elegans can be directly used for C. sp. 34 studies. We well present some of the comparative analyses of gene functions regarding the body size, germ cell formation and sex determination.
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[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
In 1998, the complete C. elegans genome sequence was published as the first metazoan genome. Since then, many metazoan genomes have been sequenced, which revealed that each animal genome contains 15,000~25,000 protein-coding genes. However, we still need to experimentally analyze when, where, and how these genes work to fully understand the genetic networks that control developmental processes. To address these issues, C. elegans early embryos are one of the best model systems, owing to the work of many years by the scientists in the worm community. Comprehensive information of the reproducible patterns of cell division and cell fate determination, sophisticated genetic techniques, and ever-improving imaging technology make this system unique such that dynamic developmental processes can be genetically dissected at a single cell level within a live embryo. In this talk, I will review some of the technical breakthroughs and how they contributed to our understanding of the early embryogenesis.
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[
International Worm Meeting,
2009]
g-tubulin mainly localizes at centrosomes and play key roles in microtubule nucleation in eukaryotic cells. In many metazoans, g-tubulin forms two types of g-tubulin complexes, termed the g-tubulin small complex (gTuSC) and the g-tubulin ring complex (gTuRC), the latter of which consists of multiple gTuSCs and additional subunits. In C. elegans, the
tbg-1 gene encodes g-tubulin (Bobinnec et al., 2000; Strome et al., 2001; Hannak et al., 2002), and
gip-1 and
gip-2 encode the homologs of the components of gTuSC, Dgrip91/GCP3/Spc98p and Dgrip84/GCP2/Spc97p, respectively (Hannak et al., 2002). However, no clear homologs for the gTuRC-specfic components (Dgrip75/GCP4, Dgrip128/GCP5, Dgrip163/GCP6, and Dgrip71WD/GCP-WD) have been identified based on the sequence similarity, thus it is unclear whether a gTuRC-like complex is present in C. elegans. To clarify the composition of the g-tubulin complexes and their function in microtubule nucleation in C. elegans, we aimed to purify g-tubulin complexes from C. elegans embryos. We established integrant lines that express the FLAG-tagged TBG-1 protein and purified the TBG-1-associating proteins by immunoprecipitation. Mass spectrometric analysis revealed that GIP-1 and GIP-2 were included in the co-purified proteins, demonstrating that g-tubulin forms a complex with GIP-1 and GIP-2 in vivo, possibly as gTuSC. We are currently analyzing other proteins co-purified with TBG-1, and will ask whether or not a gTuRC-like complex exists in C. elegans.
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[
International Worm Meeting,
2015]
Microtubules (MTs) are dynamic polymers composed of alpha- and beta-tubulin heterodimers. Most organisms have multiple tubulin isotypes encoded by different genes. In C. elegans, nine alpha-tubulins and six beta-tubulins are present. Among them, two alpha-tubulins (
tba-1 and
tba-2; hereafter called alpha1 and alpha2) and two beta-tubulins (
tbb-1 and
tbb-2; hereafter called beta1 and beta2) are the principal isotypes during embryogenesis. While the depletion of alpha1, alpha2 or beta1 does not affect viability, beta2 depletion causes abnormally active spindle movement which leads to partial embryonic lethality, implying that the two beta-tubulin isotypes distinctively contribute to the behaviors of MTs. However, how tubulin isotypes affect MT dynamics in vivo is still poorly understood. To further understand the distinct contribution of tubulin isotypes in MT dynamics in C. elegans embryos, we have generated in-frame GFP insertions in the genomic locus of beta1 or beta2 using the CRISPR-Cas9 system. Live imaging of these strains revealed that beta2 was incorporated into the spindle MTs twice as much as beta1. We next measured the parameters of the MT dynamics in embryos lacking either beta1 or beta2, using a growing MT plus-end marker GFP::EBP-2. In embryos lacking beta1, both the growth rate and the catastrophe frequency of MTs were slightly decreased, resulting in the equivalent length of MTs with the wild type despite the alteration of MT dynamics. On the other hand, in embryos lacking beta2, the growth rate was severely decreased and the catastrophe frequency was increased, resulting in shorter MTs that lead to abnormally active spindle movement. The decreased growth rate (and resulting partial embryonic lethality) by beta2 depletion was enhanced by alpha1 depletion, whereas it was partially suppressed by alpha2 depletion. These results indicate that different tubulin isotypes-and different combinations of alphabeta-heterodimers-distinctively affect MT dynamics.
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[
International Worm Meeting,
2013]
In animal mitotic cells, spindles are formed mainly from microtubules assembled at centrosomes, with an additional contribution of microtubules assembled around condensed chromosomes. In contrast, female meiotic spindles are assembled independently of centrosomes, because centrosomes are eliminated during oogenesis. In C. elegans, the formation of female meiotic spindle is initiated inside the nucleus of mature oocytes, and then the spindle undergoes dynamic structural changes during meiosis progression. In late anaphase, the meiotic spindle is re-organized and microtubules are formed between chromosomes to segregate them. It has been reported that the gamma-tubulin complex, the major microtubule nucleator during mitosis, is dispensable for the formation of female meiotic spindles in C. elegans.
We previously reported that, in mitosis of C. elegans zygotes, the assembly of microtubules around condensed chromosomes requires the kinase-inactive form of Aurora A (AIR-1) but not gamma-tubulin (TBG-1). Here, we examined the role of AIR-1 in the assembly of microtubules composing female meiotic spindles. Immunostaining revealed that the AIR-1 protein localized to meiotic spindle microtubules and cytoplasmic microtubules in zygotes undergoing meiotic divisions, whereas the localization of phosphorylated (kinase-active) AIR-1 is limited to microtubules between chromosomes in meiotic anaphase.
air-1(RNAi) did not inhibit the initial stage of meiotic spindle formation, but in late anaphase the reorganization of microtubules between chromosomes was severely affected, resulting in chromosome segregation defects. In addition,
air-1(RNAi) significantly reduced the cytoplasmic microtubules at late anaphase of female meiosis. Live-imaging and photo-bleaching experiments revealed that cytoplasmic microtubules flowed into the meiotic spindle in which microtubules were rapidly turned over. Taken together, we propose that cytoplasmic microtubules stabilized and/or formed dependently on AIR-1 may contribute to the microtubule reorganization of the female meiotic spindle in late anaphase.
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[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
P granules are large ribonucleoprotein (RNP) complexes present in the germ lineage cells throughout the development of C. elegans, which are thought to play important roles in germ cell specification and differentiation. Many components of P granules have been identified. However, how they interact to form P granules is unknown. ?We established a granule formation assay using mammalian cells, and found that PGL proteins (PGL-1 and PGL-3) formed globular granules autonomously and recruited some other P granule components. In addition, PGL proteins formed globular granules when they were expressed in the somatic cells of C. elegans embryos and adults. Furthermore, when PGL proteins were depleted in the early embryos, other P granule components, GLH-1 and POS-1, were dispersed into the cytoplasm, suggesting that PGL proteins indeed function as the scaffold t o assemble P granules. ?To further investigate the roles of PGL proteins in P granule formation, we analyzed the functional domains of PGL-3 in mammalian cells and in C. elegans embryos. We found that RGG box of PGL proteins, which binds to RNA, is dispensable for granule formation, but required for recruitment of RNA and some other P granule components. We also found that 159-318aa is involved in self-interaction. Therefore, we propose that PGL-3 has two functional domains, one for self-interaction and another for RNA/RNP binding via RGG box, and that both functions are important for P granule formation. ?Live imaging of P granules indicated that P granules are rapidly disassembled or degraded in the soma in early embryogenesis. We found that PGL-3 was phosphorylated in vivo, and detected several candidate sites for phosphorylation in PGL-3. We speculate that these potential phosphorylation sites could be important for the regulation of stability of P granules.
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[
International Worm Meeting,
2011]
Aurora A kinase is one of the key regulators of mitotic events including mitotic entry, centrosome maturation and spindle bipolarity. We previously found that C. elegans Aurora A (AIR-1) is responsible for the assembly of g-tubulin-independent microtubules in early embryos (1); however, the mechanism by which AIR-1 contributes to microtubule assembly during mitosis has been unclear.
In other organisms, condensed chromatin induces spindle microtubule assembly by creating a local environment that is favorable for microtubules nucleation and stabilization. We found by live imaging and RNAi in C. elegans early embryos that AIR-1 has a crucial role in the assembly of chromatin-stimulated microtubules that is independent of the g-tubulin complex. Surprisingly, the kinase activity of AIR-1 is dispensable for this process. Whereas the kinase-inactive form of AIR-1 was detected along the microtubules as well as on centrosomes, the kinase-active form of AIR-1-recognized with anti-phospho-AIR-1-was restricted to centrosomes. Furthermore, embryos expressing kinase-inactive forms of AIR-1 (in the absence of endogenous AIR-1) were able to assemble chromatin-stimulated microtubules, although they were defective in centrosome maturation.
TPXL-1 is a potent activator of the AIR-1 kinase activity in vitro (2), but we found that AIR-1 at centrosomes in
tpxl-1(RNAi) embryos was kinase-active, suggesting that TPXL-1 is not the major AIR-1 kinase activator in vivo. Although AIR-1 does not localize along microtubules in
tpxl-1(RNAi) embryos (2), short chromatin-stimulated microtubules were still formed by the contribution of AIR-1. Thus, we speculate that AIR-1 can mediate the formation of chromatin-stimulated microtubules without localizing along them, and that its localization along microtubules via TPXL-1 enhances its microtubule stabilizing effect.
Taken together, we propose that AIR-1 has a kinase-dependent role at centrosomes and a kinase-independent role for stabilizing spindle microtubules, and that coordination of these two roles is crucial for the assembly of mitotic spindles.
1. Motegi et al. Dev Cell 10, 509-20 (2006)
2. Ozlu et al. Dev Cell 9, 237-48 (2005).
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[
International Worm Meeting,
2009]
In many organisms, germ cells have regions of specialized cytoplasm, generally referred to as "germ granules". They are large RNA-enriched nonmembranous organelles, and believed to play roles in germ cell specification and differentiation. Germ granules in C. elegans are called P granules, which are specifically segregated into the germ lineage during early embryogenesis and present in germ cells throughout development. Although ~20 protein (mostly RNA binding proteins) and several RNA components of P granules have been identified, it is still unclear how they are assembled and segregated into germ cells. To dissect the assembly mechanism of P granules, we have developed an assay system to test the granule formation ability of each P granule component using cultured mammalian cells. We have found that, among the 14 P granule components examined, only PGL-1 and PGL-3 proteins have the ability to form cytoplasmic granules autonomously in mammalian cells. Interestingly, these granules formed by PGL proteins exhibited a layered architecture and encapsulated endogenous RNA and poly(A) binding protein, and some co-expressed P granule components. A high resolution microscopy revealed that some P granules in C. elegans embryos and adult gonads also show a similar layered architecture. When PGL proteins were ectopically expressed in somatic cells in adult worms, they formed cytoplasmic granules, confirming that PGL proteins can form granules in the absence of other germline-specific components. Structure-function analyses of the PGL-3 protein in mammalian cells and C. elegans embryos indicated that RGG box, an RNA-binding motif, at the C-terminus of the protein is dispensable for granule formation, but required for recruiting RNA and other P granule components. Based on these results, we propose that the ability of the PGL proteins to autonomously form RNP granules play a crucial role in the assembly and architecture of P granules in C. elegans. We speculate that the layered architecture formed by PGL proteins may be advantageous to sequester specific maternal mRNAs that are to be delivered to and/or stabilized in the germ lineage.
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[
International Worm Meeting,
2009]
P granules are ribonucleoprotein (RNP) complexes and thought to play important roles in germ cell development. Although many components of P granules have been identified, how they interact to form P granules is unknown. To elucidate the mechanism of P granule formation, we recently established a granule assembly assay using cultured mammalian cells (CHO cells). We found that, among the 14 P granule components tested, only PGL-1 and PGL-3 autonomously formed granules, whereas other P granule components scattered throughout the cytoplasm in CHO cells. The PGL granules formed in CHO cells recruited other P granule components when co-expressed, as well as endogenous RNA and poly(A) binding protein. Thus, PGL proteins appeared to play crucial roles in RNP granule formation. To further investigate the roles of PGL proteins, we conducted a structure-function analysis of PGL-3 in CHO cells and C. elegans embryos. PGL-1 and PGL-3 contain an RNA-binding motif, RGG box, in their C termini. We found that RGG box of PGL-3 was dispensable for granule formation, but essential for recruiting endogenous RNA and other P granule components into the PGL granules in mammalian cells. We further found that 161-479aa of PGL-3 is sufficient for granule formation, and that 159-318aa is required for self-interaction. These functional domains of PGL-3 identified in mammalian cells were next examined in C. elegans embryo, by expressing GFP-tagged PGL-3 variants. We confirmed that 159-318aa was required for the targeting of PGL-3 to P granules, whereas RGG box was dispensable. Our results suggest that PGL-3 contains distinct domains for self-interaction/granule formation and RNA binding, both of which cooperatively contribute to P granule formation.
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[
International Worm Meeting,
2021]
Caenorhabditis inopinata, the sibling species of C. elegans, recently discovered in Ryukyu islands Japan, has a distinct lifestyle from other known Caenorhabditis species. The gonochoristic species inhabits in syconia of a fig tree (Ficus septica) and uses the pollinator wasp (Ceratosolen sp.) as a vector to move from old to new syconia. We isolated C. inopinata from fig syconia collected in seven Ryukyu islands and Taiwan (distance in 730 km), and established over 20 single female lines. Phylogenetic analyses of those lines revealed that C. inopinata can be separated largely into two geographical groups (Okinawa and Ishigaki groups). Although we couldn't identify clear morphological differences, the genetic distance of the two groups calculated by genome comparisons was relatively large as an intra-species diversity. We therefore sought to clarify mating and reproductive compatibilities between the two geographical groups. Using three C. inopinata lines each from Okinawa and Ishigaki groups, respectively, we observed mating behaviours, embryo developments and reproductions for all line combinations and directions (male - female). Mating behaviours were observed in all combinations though sexual attraction levels seem lower in inter-group than intra-group combinations. However, offspring (F1) numbers were smaller in inter-group combination, especially incomplete embryo developments and almost no offspring were observed at Okinawa male and Ishigaki female combination. These results suggest that speciation in C. inopinata has possibly been accelerated by the characteristic lifestyle and the island effect in this small area.