The sister chromatid cohesion complex of Saccharomyces cerevisiae is composed of proteins termed cohesins. The complex forms a ring structure that entraps sister DNAs, probably following replication. The mechanism of cohesion is universal and the proteins participating in this process are evolutionarily highly conserved. We investigated the Irr1p/Scc3p cohesin subunit, an under-studied protein. We show that the presence of a mutated copy of IRR1 gene, encoding the F658G substitution in Irr1p, changes the sensitivity of the heterozygous irr1-1/IRR1 diploid to cell wall-affecting compounds. Microscopic images indicate that chitin distribution in the mutant cell wall is affected, although the biochemical composition of the cell wall is not drastically changed. This observation suggests that irr1-1 mutation in heterozygous state may influence the cell wall integrity and indicates a possible link between mechanisms regulating the cell wall biosynthesis, nuclear migration and chromosome segregation.

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Although the consequences of Ras activation have been studied extensively in the context of oncogenesis, its regulation in physiological modes of signal transduction is not well understood. A fluorescent indicator, Raichu-Ras, was fused to the C-terminal hypervariable regions of H-Ras and K-Ras to create indicators for Ras activation within caveolae/rafts (Raichu-tH) and non-raft domains (Raichu-tK) of the plasma membrane, respectively. Raichu-tH was also found abundantly in endomembranes. To monitor Ras activation with high spatial resolution, it is imperative to observe sectioned images of the signals. We have developed a wide-field fluorescence microscope equipped with a digital micromirror device (DMD) to acquire optically sectioned images using fringe projection. This system provides reliable signals from fluorescence resonance energy transfer (FRET) between cyan and yellow mutants of green fluorescent protein. We have used this system to demonstrate that, upon stimulation with growth factors, the two indicators are activated in spatially and temporally unique patterns.

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A basic feature of the movement of eukaryotic flagella is oscillation. Although flagellar oscillation is thought to be regulated by a self-regulatory feedback system including the mechanical signal of bending itself, the mechanism regulating the dynein motile activity to produce oscillation is not well understood. To elucidate the mechanism, we developed a new experimental system which allowed us to analyze the conditions necessary for the induction of oscillation. When a mechanical signal of bending or a pulse was applied by micromanipulation to a demembranated motionless sea urchin sperm flagellar axoneme at very low ATP concentrations (1–3 μM), a localized pair of bends was induced. The bend formation was often followed by further responses including propagation of the distal bend of paired bends, growth and propagation of the paired bends, and cyclical beating. The beating was induced at 2.0 μM or higher concentrations of ATP, but appeared even at 1.5 μM ATP if a few μM of ADP was also present. When the proximal half of a flagellum was attached to a microneedle, beating could not be induced in the distal free region at 2 μM ATP. These results suggest that mechanical signal is involved in the mechanism regulating the motile activity of dynein to produce oscillation. Our results also showed that the presence of a small amount of ADP and the axial difference along the flagellum are factors essential for the induction of flagellar oscillation.

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Polar body formation is an extremely unequal cell division. In order to understand the mechanism of polar body formation, morphological changes at the animal pole were investigated in living oocytes of the starfish, Asterina pectinifera, and the amounts of cortical actin filaments were quantitatively estimated after staining the maturing oocytes with fluorescently-labeled phallotoxins using a computer and image-processing software. Formation of a bulge, which is presumed to become a polar body, and the anaphase separation of chromosomes occurred simultaneously. When the bulge became large, one group of chromatids moved into the bulge. The dividing furrow then formed and finally a polar body formed. Just at the time of bulge formation, the intensity of the fluorescence produced by the actin filaments at the top of the animal pole began to decrease, and subsequently the intensity at the top fell to half of the original value. On the other hand, the fluorescence intensity at the base of the bulge increased gradually. This actin accumulation at the base created a dividing furrow around the top of the animal pole as the bulge grew. Even when the polar body formation was inhibited mechanically, a similar pattern of actin deficiency and accumulation in the cortex near the animal pole was observed. This indicates that such regulation of filamentous actin can take place without bulging. Therefore, polar body formation is initiated by the bulging of the cortex weakened by actin deficiency and followed by contraction of the base of the bulge reinforced by actin accumulation.

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Arginine rich, mutated in early stage of tumors (ARMET) was first identified as a human gene highly mutated in a variety of cancers. However, little is known about the characteristics of the ARMET protein and its expression. We identified ARMET as a gene upregulated by endoplasmic reticulum (ER) stress. Here, we show that the mouse homologue of ARMET is an 18-kDa soluble ER protein that is mature after cleavage of a signal sequence and has four intramolecular disulfide bonds, including two in CXXC sequences. ER stress stimulated ARMET expression, and the expression patterns of ARMET mRNA and protein in mouse tissues were similar to those of Grp78, an Hsp70-family protein required for quality control of proteins in the ER. A reporter gene assay using a mouse ARMET promoter revealed that the unfolded protein response of the ARMET gene is regulated by an ERSE-II element whose sequence is identical to that of the HERP gene. ARMET is the second fully characterized ERSE-II-dependent gene and likely contributes to quality control of proteins in the ER.

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The effects of heavy ion microbeams on muscle fibers isolated from mouse skeletal muscles were examined by electron microscopy. The plasma membranes of heavy ion beam-irradiated areas of muscle fibers showed irregular protrusions and invaginations. In the cytoplasm, an irregular distribution of microfilaments was found near the plasma membrane. Sarcoplasmic reticula in the irradiated regions showed a distended appearance with flocculent material within the lumen. These changes were seen as early as 2 min after irradiation, and persisted until as late as 22 min after irradiation. Many autophagic vacuoles could be seen at 7 min after irradiation. At 22 min, the vacuoles became more prominent and showed more variety. These observations suggest that heavy ion beam irradiation causes disruption of the cellular architecture and the autophagy is involved in removal of this disruption.

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Three subtypes of HP1, a conserved non-histone chromosomal protein enriched in heterochromatin, have been identified in humans, HP1α, β and γ. In the present study, we utilized a Drosophila system to characterize human HP1 functions. Over-expression of HP1β in eye imaginal discs caused abnormally patterned eyes, with reduced numbers of ommatidia, and over-expression of HP1γ in wing imaginal discs caused abnormal wings, in which L4 veins were gapped. These phenotypes were specific to the HP1 subtypes and appear to reflect suppressed gene expression. To determine the molecular domains of HP1 required for each specific phenotype, we constructed a series of chimeric molecules with HP1β and HP1γ. Our data show that the C-terminal chromo shadow domain (CSD) of HP1γ is necessary for HP1γ-type phenotype, whereas for the HP1β-type phenotype both the chromo domain and the CSD are required. These results suggest human HP1 subtypes use different domains to suppress gene expression in Drosophila cells.

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To analyze the functional differences of the insulin receptor substrate (IRS) family, the N-terminal fragments containing the pleckstrin homology (PH) domains and the phosphotyrosine-binding (PTB) domains of IRS (IRS-N) proteins, as well as intact IRS molecules, were expressed in Cos-1 cells, and insulin-induced tyrosine phosphorylation and subcellular distribution of IRS proteins were analyzed. In contrast to the distinct affinities toward phosphoinositides, these IRS-N fragments non-selectively inhibited insulin-induced tyrosine phosphorylation of IRS-1, IRS-2 and IRS-3, among which IRS3-N was most effective. The mutations of IRS-1 disrupting all the phosphoinositide-binding sites in both the PH and PTB domains significantly but not completely suppressed tyrosine phosphorylation of IRS-1, which was further inhibited by coexpression of all the IRS-N proteins examined. In contrast, the N-terminal PH domain-interacting region (PHIP-N) of PH-interacting protein (PHIP) did not impair tyrosine phosphorylation of either IRS molecule. The analysis using confocal microscopy also demonstrated that all the IRS-N proteins, but not PHIP-N, suppressed targeting of IRS-1 to the plasma membrane in response to insulin. Moreover, the phosphoinositide affinity-disrupting mutations of IRS-1 significantly impaired but did not completely abrogate the insulin-induced translocation of IRS-1 to the plasma membrane, which was further suppressed by IRS1-N overexpression. These findings suggest that both insulin-induced tyrosine phosphorylation and the cell surface targeting of IRS proteins may be regulated in a similar manner through a target molecule common to the members of the IRS family, and distinct from phosphoinositides or PHIP.

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