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Volume 30 (2005)
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Volume 30, Issue 2
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Taro Nakamura, Jun Kashiwazaki, Chikashi Shimoda2005 Volume 30 Issue 2 Pages 15-24
Published: 2005
Released on J-STAGE: November 02, 2005
JOURNAL FREE ACCESS FULL-TEXT HTMLThe soluble NSF attachment protein 25 (SNAP-25) is a component of the SNARE complex that is essential for regulated exocytosis in diverse cell types. Here, we identified a fission yeast SNAP-25 homologue, SpSec9. The sec9+ gene was essential for vegetative growth. sec9 mRNA was detected in vegetative cells and further increased during sporulation. This increase during sporulation was dependent on Mei4, a meiosis-specific transcription factor. A sporulation-deficient sec9 mutant was isolated by random PCR mutagenesis (sec9-10). The sec9-10 mutant also exhibited temperature sensitivity for growth and cell division was found to arrest before completion of cell separation at restrictive temperatures. In sec9-10 cells, the forespore membrane was normally initiated near spindle pole bodies during meiosis II. However, subsequent extension of the membrane was severely impaired. These results indicate that SpSec9 plays an important role both in cytokinesis and in sporulation.
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Yoshitaka Fukunaga, Huijie Liu, Masayuki Shimizu, Satoshi Komiya, Mich ...2005 Volume 30 Issue 2 Pages 25-34
Published: 2005
Released on J-STAGE: December 16, 2005
JOURNAL FREE ACCESS FULL-TEXT HTMLF9 teratocarcinoma cells in which β-catenin and/or plakoglobin genes are knocked-out were generated and investigated in an effort to define the role of β-catenin and plakoglobin in cell adhesion. Loss of β-catenin expression only did not affect cadherin-mediated cell adhesion activity. Loss of both β-catenin and plakoglobin expression, however, severely affected the strong cell adhesion activity of cadherin. In β-catenin-deficient cells, the amount of plakoglobin associated with E-cadherin dramatically increased. In β-catenin/plakoglobin-deficient cells, the level of E-cadherin and α-catenin markedly decreased. In these cells, E-cadherin formed large aggregates in cytoplasm and membrane localization of α-catenin was barely detected. These data confirmed that β-catenin or plakoglobin is required for α-catenin to form complex with E-cadherin. It was also demonstrated that plakoglobin can compensate for the absence of β-catenin. Moreover it was suggested that β-catenin or plakoglobin is required not only for the cell adhesion activity but also for the stable expression and cell surface localization of E-cadherin.
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Rina Kimura, Ryo Iwamoto, Eisuke Mekada2005 Volume 30 Issue 2 Pages 35-42
Published: 2005
Released on J-STAGE: December 16, 2005
JOURNAL FREE ACCESS FULL-TEXT HTMLHeparin-binding EGF-like growth factor (HB-EGF), a member of the EGF-family, is thought to be important for keratinocyte functions. HB-EGF is first synthesized as a membrane-anchored form, and its soluble form is released by ectodomain shedding. Here we investigate the role of HB-EGF in epidermal hyperplasia induced by all-trans retinoic acid (tRA) treatment. HB-EGF is normally expressed in epidermis of normal adult mice at very low levels, but topical tRA treatment results in epidermal hyperplasia, concomitant with the strong induction of HB-EGF expression in the suprabasal layer. tRA-induced epidermal hyperplasia was reduced both in the keratinocyte-specific HB-EGF null mice (K5-HBdel/del) and knock-in mice expressing the uncleavable mutant form of HB-EGF (HBuc/uc), as compared with wild-type HB-EGF knock-in mice (HBlox/lox). Among ErbB tyrosine kinase receptors, EGF receptor (EGFR) and ErbB2 were selectively activated by tRA treatment in skin from wild-type mice, while the activation of these ErbB receptors was significantly reduced in the skin of HB-EGF null mice. These results indicate that expression of HB-EGF and generation of its soluble form, followed by activation of EGFR and ErbB2, are pivotal processes in tRA-induced epidermal hyperplasia.
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Jun Kashiwazaki, Taro Nakamura, Tomoko Iwaki, Kaoru Takegawa, Chikashi ...2005 Volume 30 Issue 2 Pages 43-49
Published: 2005
Released on J-STAGE: December 16, 2005
JOURNAL FREE ACCESS FULL-TEXT HTMLYpt7p, a fission yeast (Schizosaccharomyces pombe) homologue of Rab7 GTPase, mediates fusion of endosomes to vacuoles and homotypic vacuole fusion. Here, we report that Ypt7p plays important roles in sporulation. Most ypt7Δ asci produced less than four spores, which were apparently immature and germinated at low frequency. Furthermore, ypt7Δ cells were defective in development of the forespore membranes. Vacuoles in sporulating cells were found to undergo extensive homotypic vacuole fusion to form a few large compartments occupying the entire cytoplasm of asci. This extensive vacuole fusion depended on Ypt7p.
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Ryoji Igarashi, Masaya Suzuki, Satoru Nogami, Yoshikazu Ohya2005 Volume 30 Issue 2 Pages 57-67
Published: 2005
Released on J-STAGE: January 17, 2006
JOURNAL FREE ACCESS FULL-TEXT HTMLThe dynactin complex is one of the components required for the regulation of the cell wall integrity checkpoint, which ensures the completion of cell wall remodeling before mitosis. The core of the dynactin complex is a backbone filament composed of monomers of an actin-related protein, Arp1, which is also involved in nuclear migration. To examine the molecular basis for the dual functions of the dynactin core subunit Arp1p in yeast, we constructed 32 mutated arp1 alleles. We assessed the effects of the mutations on cell wall integrity checkpoint and nuclear migration functions and identified four categories of mutants: 1) those showing no change from the wild type; 2) those resulting in a defective cell wall integrity checkpoint but normal nuclear migration; 3) those with a normal cell wall integrity checkpoint but defective nuclear migration; and 4) those defective in both the cell wall integrity checkpoint and nuclear migration functions. Our results show a separation of the two functions in the molecular structure of Arp1p and indicate that a local surface region of Arp1p is important in maintaining the cell wall integrity checkpoint function.
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Eri Takeda, Miki Hieda, Jun Katahira, Yoshihiro Yoneda2005 Volume 30 Issue 2 Pages 69-80
Published: 2005
Released on J-STAGE: January 23, 2006
JOURNAL FREE ACCESS FULL-TEXT HTMLRan is a nuclear Ras-like GTPase that is required for various nuclear events including the bi-directional transport of proteins and ribonucleoproteins through the nuclear pore complex, spindle formation, and reassembly of the nuclear envelope. One of the key regulators of Ran is RanGAP1, a Ran specific GTPase activating protein. The question of whether a mechanism exists for controlling nucleocytoplasmic transport through the regulation of RanGAP1 activity continues to be debated. Here we show that RanGAP1 is phosphorylated in vivo and in vitro. Serine-358 (358S) was identified as the major phosphorylation site, by MALDI-TOF-MS spectrometry. Site directed mutagenesis at this position abolished the phosphorylation. Experiments using purified recombinant kinase and specific inhibitors such as DRB and apigenin strongly suggest that casein kinase II (CK2) is the responsible kinase. Although the phosphorylation of 358S of RanGAP1 did not significantly alter its GAP activity, the phosphorylated wild type RanGAP1, but not a mutant harboring a mutation at the phosphorylation site 358S, efficiently formed a stable ternary complex with Ran and RanBP1 in vivo, suggesting that the 358S phosphorylation of RanGAP1 affects the Ran system.
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Kayoko Tsuda, Atsuo Amano, Kyohei Umebayashi, Hiroaki Inaba, Ichiro Na ...2005 Volume 30 Issue 2 Pages 81-91
Published: 2005
Released on J-STAGE: January 23, 2006
JOURNAL FREE ACCESS FULL-TEXT HTMLPorphyromonas gingivalis is one of the causative agents of adult periodontitis, and has been reported to be internalized by nonphagocytic epithelial cells. However, the mechanism for the internalization remains unclear. In the present study, we addressed this issue using fluorescent beads coated with bacterial membrane vesicles (MVs) that retain surface components of P. gingivalis. We established an assay system in which we could easily quantify the bead internalization to cells. MVs-coated beads were internalized by HeLa cells in kinetics similar to that of living bacteria. The internalization depended on dynamin but not clathrin. The beads were internalized through the actin-mediated pathway that is controlled by phosphatidylinositol (PI) 3-kinase. The dynamics of microtubule assembly and disassembly was also required. Further, the treatment of cells with cholesterol-binding reagents significantly inhibited bead internalization, and the internalized beads were apparently colocalized with ganglioside GM1 and caveolin-1, which suggest the involvement of the lipid raft in the process. These results suggest that P. gingivalis accomplishes its internalization utilizing membrane lipid raft and cytoskeletal functions of the target cells.
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Dai Shiba, Tetsuro Takamatsu, Takahiko Yokoyama2005 Volume 30 Issue 2 Pages 93-100
Published: 2005
Released on J-STAGE: February 11, 2006
JOURNAL FREE ACCESS FULL-TEXT HTML
Supplementary materialPrimary cilia are hypothesized to act as a mechanical sensor to detect renal tubular fluid flow. Anomalous structure of primary cilia and/or impairment of increases in intracellular Ca2+ concentration in response to fluid flow are thought to result in renal cyst formation in conditional kif3a knockout, Tg737 and pkd1/pkd2 mutant mice. The mutant inv/inv mouse develops multiple renal cysts like kif3a, Tg737 and pkd1/pkd2 mutants. Inv proteins have been shown to be localized in the renal primary cilia, but response of inv/inv cilia to fluid stress has not been examined. In the present study, we examined the mechanical response of primary cilia to physiological fluid flow using a video microscope, as well as intracellular Ca2+ increases in renal epithelial cells from normal and inv/inv mice in response to flow stress. Percentages of ciliated cells and the length of primary cilia were not significantly different between primary renal cell cultures from normal and inv/inv mutant mice. Localization of inv protein was restricted to the base of primary cilia even under flow stress. Inv/inv mutant cells had similar bending mechanics of primary cilia in response to physiological fluid flow compared to normal cells. Furthermore, no difference was found in intracellular Ca2+ increases in response to physiological fluid flow between normal and inv/inv mutant cells. Our present study suggests that the function of the inv protein is distinct from polaris (the Tg737 gene product), polycystins (pkd1 and pkd2 gene products).
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REVIEW
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Takashi Miki, Keizo Takano, Yoshihiro Yoneda2005 Volume 30 Issue 2 Pages 51-56
Published: 2005
Released on J-STAGE: December 26, 2005
JOURNAL FREE ACCESS FULL-TEXT HTMLThe localization of mRNA in neuronal dendrites plays a role in both locally and temporally regulated protein synthesis, which is required for certain forms of synaptic plasticity. RNA granules constitute a dendritic mRNA transport machinery in neurons, which move along microtubules. RNA granules contain densely packed clusters of ribosomes, but lack some factors that are required for translation, suggesting that they are translationally incompetent. Recently some of the components of RNA granules have been identified, and their functions are in the process of being examined, in attempts to better understand the properties of RNA granules. Mammalian Staufen, a double-stranded RNA binding protein, is a component of RNA granules. Staufen is localized in the somatodendritic domain of neurons, and plays an important role in dendritic mRNA targeting. Recently, one of the mammalian homologs of Staufen, Staufen2 (Stau2), was shown to shuttle between the nucleus and the cytoplasm. This finding suggests the possibility that Stau2 binds RNA in the nucleus and that this ribonucleoprotein particle is transported from the nucleus to RNA granules in the cytoplasm. A closer study of this process might provide a clue to the mechanism by which RNA granules are formed.
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