Sugimoto A (2009) F1000 Biol Rep "Clearance of germ granules in the soma."

Sugimoto A

[F1000 Biol Rep, 2009]

Germ granules are ribonucleoprotein complexes specifically segregated into germ cell lineages in diverse organisms. Recent studies indicate that multiple mechanisms are involved in the clearance of germ granules and their components in somatic cells in Caenorhabditis elegans embryos.

Sugimoto A et al. "Death signalling in C. elegans and activation mechanisms of caspases."

Masayuki M, Sugimoto A

Cell proliferation and cell death are two opposing sides of the same coin. The development and homeostasis of multicellular organisms requires the accurate control of both processes. The death that occurs normally as a process of development is called 'programmed cell death' or 'apoptosis'. The phenomenon of developmentally programmed cell death has been known for more than 100 years, but it is only in the last decade that its molecular mechanisms have begun to be uncovered. The nematode Caenorhabditis elegans is one of the few experimental systems in which a genetic approach is available to dissect the processes of programmed cell death, and in fact, findings from studies using C. elegans have already played a very important role in elucidating these mechanisms. Here, we summarise the progress to date in understanding how cell death is controlled in C. elegans, and review the main machinery of programmed cell death/apoptosis with respect to its evolutionary conservation between C. elegans and other species.

Sugimoto A et al. (1994) Worm Breeder's Gazette "Characterized cDNA Clones in High-Density Grids"

Kohara Y, Sugimoto A

[Worm Breeder's Gazette, 1994]

We are making sets of cDNA filters which are blotted with high-density grids of plaques of the some 4,400 cDNA clones most of which has been tag-sequenced as described above. The set consists of 3 nylon membrane filters (12 x 8.5 cm) on each of which cDNA clones from 15 or 16 of 96 well plate stocks are gridded as below using a home-made apparatus. We are intended to make the filters available to the worm community. Anyone who are interested in trying the filters, send your request to us at (e-mail) ykohara@ddbj.nig.ac.jp or (Fax) +81-559-75-6240.

Sugimoto A et al. (2000) Japanese Worm Meeting "RNAi Screening with a non-redundant cDNA set"

Yamamoto M, Maeda I, Sugimoto A, Kohara Y

[Japanese Worm Meeting, 2000]

We have established a systematic reverse-genetic screening method based on phenotypes, to identify genes involved in specific developmental processes. In this method, we have combined RNAi and cDNA project. Namely, we use a tag-sequenced, non-redundant cDNA set as templates for preparing dsRNA and perform RNAi systematically. To introduce dsRNA into worms, we use the 'soaking method' instead of microinjection, so that we can handle multiple samples concurrently. The soaking method had been known to be less potent, but we optimized the condition and achieved enough efficiency and sensitivity to perform screening (Maeda et al., 1999 International Worm Meeting #565). In this method, four L4 hermaphrodites are soaked in each RNA solution derived from single cDNA clone, then incubated for 24 hours. The worms are then recovered to a new plate and phenotypes of both P0s and F1 progeny are observed. All steps of RNA synthesis, purification and worm soaking are performed in 96-well format. To date, we examined 2064 RNA species. Of those, 0.9% (19/2064) caused sterility of F1s and 14.3% (295/2064) caused embryonic lethality. In total, about 28% (573/2064) of RNA species caused detectable phenotypes. We are now screening at the rate of 200 clones/month. Since we are interested in germ line development and differentiation, we are currently focusing on RNA species that cause sterility with no somatic phenotypes. Among such RNA species, various kinds of translational regulators were included. This is consistent with the fact that translational regulation plays an important role in germ line development. The sterile-only RNA species also included several known genes which have never been reported to be involved in germ line development. We plan to make RNAi data available to the public and report all detectable phenotypes at the NEXTDB WWW server at NIG. We will present the result and the status of our screening.

Sugimoto A et al. (2000) Journal of Reproduction and Development "Regulation of gametogenesis and meiosis in the nematode C. elegans."

Sugimoto A, Karashima T, Yamamoto M

[Journal of Reproduction and Development, 2000]

Gametogenesis in multicellular organisms is a complex process in which specialized cellular differentiation and the meiotic cell cycle are coordinated to produce oocytes and sperm. The DAZ (Deleted in Axoospermia) gene family provides one of the few lines of evidence that argue for evolutionary conservation of gametogenesis at the molecular level.

Sugimoto A (2004) Differentiation "High-throughput RNAi in Caenorhabditis elegans: genome-wide screens and functional genomics."

Sugimoto A

[Differentiation, 2004]

The phenomenon of RNA-mediated interference (RNAi) was first discovered in the nematode Caenorhabditis elegans, in which introduction of double-stranded RNA causes specific inactivation of genes with corresponding sequences. Technical advances in RNAi methodology and the availability of the complete genome sequence have enabled the high-throughput, genome-wide RNAi analysis of this organism. Several groups have used large-scale RNAi to systematically examine every C. elegans gene for knock-down phenotypes, providing basal information to be mined in more detailed studies. Now, in addition to functional genomic RNAi analyses, high-throughput RNAi is also routinely used for rapid, genome-wide screens for genes involved in specific biological processes. The integration of high-throughput RNAi experiments with other large-scale data, such as DNA microarrays and protein-protein interaction maps, enhances the speed and reliability of such screens. The accumulation of RNAi phenotype data dramatically accelerates our understanding of this organism

Sugimoto A et al. (2000) Japanese Worm Meeting "C. elegans homologue of protein phosphatase 4 is required in spermatogenesis"

Yamamoto M, Sugimoto A, Sumiyoshi E

[Japanese Worm Meeting, 2000]

Protein phosphatase 4 (PP4) is a ser/thr protein phosphatase, which is known to localize to centrosomes. Analysis using a fly mutant implicated that PP4 is required for the nucleation of microtubules during mitosis. However, its function in other organisms is not yet understood. In C.elegans, two possible homologues of PP4, namely Y75B8A.30 and Y49E10.3, have been identified by the genome project. Here we report characterization of Y75B8A.30. To understand the function of Y75B8A.30, we performed RNAi for it. Although the penetrance varied among injections, RNAi for Y75B8A.30 at the highest efficiency caused 25% embryonic lethality at F1 and nearly 100% embryonic lethality at F2. Y75B8A.30 (RNAi) F2 embryos showed a multi polar spindle with two male pronuclei at 1-cell stage. We suspected that an extra male pronucleus might result from a defect in spermatogenesis. To confirm this, we examined the spermatogenesis of the F1 progeny. Observation with Nomarski optics and with DAPI staining revealed that F1 spermatids often contained multiple nuclei. We also found that the nuclei of secondary spermatocytes of F1 worms were not properly separated, indicating that Y75B8A.30 is required for proper chromosome segregation in sperm meiosis. To see whether the multi polar spindle is a result of the defect in sperm, F1 males were crossed to rde-1 hermaphrodites, which is resistant to RNAi. 5/16 of cross progeny showed multi polar spindles at 1-cell stage, indicating that multi polar spindles are indeed caused by the defect of sperm at least in part. Thus, we conclude that PP4 is required for the segregation of chromosomes and centrosomes during spermatogenesis. To see whether Y75B8A.30 function is required in embryogenesis, we crossed Y75B8A.30 (RNAi) F1 hermaphrodites with wild type male. Although wild type sperm were supplied in this cross, many cross progenies died during embryogenesis, showing that Y75B8A.30 is required for embryogenesis. To know the possible role of Y75B8A.30 in mitosis, we stained F2 embryos with anti-tubulin antibody and DAPI. Some fraction of 1-cell embryos had condensed chromosomes at the metaphase plate, with microtubules mislocalized around the nucleus, suggesting that Y75B8A.30 may be involved in the organization of microtubules during mitosis.

Sugimoto A et al. (1993) International C. elegans Meeting "Baculovirus p35 inhibits programmed cell death in C. elegans."

Sugimoto A, Friesen PD, Rothman JH

[International C. elegans Meeting, 1993]

Sugimoto A et al. (1997) International C. elegans Meeting "A DEFICIENCY SCREEN FOR REGIONS OF THE GENOME INVOLVED IN PROGRAMMED CELL DEATH"

Derry B, Kusano-Fujita A, Hozak RR, Sugimoto A, Rothman JH, Zhu JW

[International C. elegans Meeting, 1997]

To identify regions of the genome required zygotically for normal programmed cell death (PCD) in C. elegans, we screened a collection of deficiencies (Dfs) which collectively delete ~80% of the genome. Homozygous Df embryos were observed by Nomarski microscopy throughout embryogenesis and the number and appearance of cell corpses were examined to identify abnormalities in PCD. The Dfs were categorized into four classes based on their PCD phenotypes: Twenty-four Dfs were not significantly different from wild-type in number or appearance of cell corpses. In the second class, comprising 13 Dfs (8 regions), no or fewer than wild-type numbers of corpses were observed. Among these, two regions when deleted completely block PCD but not cell proliferation; one of these includes ced-3. The relevant gene in the other region is probably egl-1, in which loss-of-function mutations block PCD (Conradt and Horvitz, '95 Worm Meeting). The third class consists of 18 deficiencies (16 regions) that show higher-than-normal numbers of corpses. Some of these were further characterized and found to be defective in corpse engulfment. The final class includes three deficiencies (2 regions) that result in abnormally large corpses; this phenotype is probably attributable to premature cell division arrest. Most regions identified do not include known cell death genes, demonstrating the existence of additional zygotic genes required for normal embryonic PCD. For the regions showing interesting cell death phenotypes, we are currently narrowing down the location of the relevant genes using overlapping Dfs.

Sugimoto A et al. (2000) Japanese Worm Meeting "Characterization of a deficiency mutant that produces abnormally large cell corpses in late embryogenesis."

Sugimoto A, Yamamoto M, Furuya M, Rothman JH

[Japanese Worm Meeting, 2000]

Programmed cell death (PCD/ apoptosis) is a common cell fate in the development of multicellular organisms. To understand this process further, we screened for regions of C. elegans genome required zygotically for normal PCD, using a collection of chromosomal deficiency mutants which collectively deletes ~80% of the genome. From this screen, we found that a deficiency mutant tDf6 exhibited abnormally large corpses in the late embryogenesis. In addition, 4D recording analysis revealed that tDf6 embryos produced fewer corpses than the wild-type. In the ced-3 ; tDf6 double mutant, neither normal nor large corpse was observed, suggesting that the large corpses in tDf6 were produced by the ced-3 - dependent PCD pathway. To identify the gene responsible for these defects, we narrowed down the genomic region by examining the PCD phenotype of chromosomal deficiencies overlapping with tDf6. We then performed RNAi experiments for the ORFs in the narrowed region, and identified an ORF whose RNAi phenocopies tDf6. Some of the RNAi-treated embryos in which maternal expression of the ORF was likely to be inhibited, arrested with multinucleate blastomeres. 4D recording revealed that these embryos were defective in the late step of cytokinesis during early embryonic divisions. The cleavage furrow ingressed to a deep position, but it regressed after the daughter nuclei were separated. After the regression of the cleavage furrow, daughter nuclei started another round of the mitotic cycle but failed to complete cytokinesis again. This cycle was repeated multiple times. The ORF appeared to encode a GTPase-activating protein (GAP) for Rho-like GTPases, which was homologous to human MgcRacGAP, mouse band25, Drosophila Rotund. Rho family small G proteins are known to be responsible for the actin reorganization of cytoskelton during cytokinesis. From these results, we suspect that the large corpses in tDf6 are likely to be caused by the death of multinucleate cells generated by the cytokinetic defect in late embryogenesis.