Golgi inheritance in the primitive red alga, Cyanidioschyzon merolae

The Golgi body has important roles in modifying, sorting, and transport of proteins and lipids. Eukaryotic cells have evolved in various ways to inherit the Golgi body from mother to daughter cells, which allows the cells to function properly immediately after mitosis. Here we used Cyanidioschyzon merolae, one of the most suitable systems for studies of organelle dynamics, to investigate the inheritance of the Golgi. Two proteins, Sed5 and Got1, were used as Golgi markers. Using immunofluorescence microscopy, we demonstrated that C. merolae contains one to two Golgi bodies per cell. The Golgi body was localized to the perinuclear region during the G1 and S phases and next to the spindle poles in a microtubule-dependent manner during M phase. It was inherited together with spindle poles upon cytokinesis. These observations suggested that Golgi inheritance is dependent on microtubules in C. merolae.     

Transgenic zebrafish for ratiometric imaging of cytosolic and mitochondrial Ca response in teleost embryo

Intracellular Ca²⁺ imaging has widely been used to visualize intracellular signals, but the application in an intact animal is still limited due to difficulty of the indicator loading. In addition, the motion of the living animal produces artifacts. To investigate Ca²⁺ signaling at early embryonic stage, we established transgenic zebrafish line expressing a genetically encoded Ca²⁺ indicator, cameleon YC2.60, driven by a constitutively active promoter, hspa8. Although the embryo dynamically changes its morphology, the motion artifact could be canceled out by taking the advantage of YC2.60 as a ratiometric indicator. The transgenic zebrafish was used to visualize the propagation of cytosolic Ca²⁺ during the early embryonic stage upon fertilization and along cleavage furrow, and the rise in Ca²⁺ in the myocytes contracting spontaneously in the embryo. We also established a transgenic zebrafish line expressing YC2.60 targeted to the mitochondria. The rise in mitochondrial Ca²⁺ was rather sustained (≈2 min), which is consistent with the requirement of ATP refilling since the mitochondrial Ca²⁺ upregulates rate-limiting enzymes of Krebs cycle. This is in contrast with the transient rise in the cytosol Ca²⁺ that directly evokes the muscle contraction. These transgenic zebrafish lines are expected to serve as useful tools further Ca²⁺ imaging in vivo.     

Variation of the Virus-Related Elements within Syntenic Genomes of the Hyperthermophilic Archaeon Aeropyrum

The increasing number of genome sequences of archaea and bacteria show their adaptation to different environmental conditions at the genomic level. Aeropyrum spp. are aerobic and hyperthermophilic archaea. Aeropyrum camini was isolated from a deep-sea hydrothermal vent, and Aeropyrum pernix was isolated from a coastal solfataric vent. To investigate the adaptation strategy in each habitat, we compared the genomes of the two species. Shared genome features were a small genome size, a high GC content, and a large portion of orthologous genes (86 to 88%). The genomes also showed high synteny. These shared features may have been derived from the small number of mobile genetic elements and the lack of a RecBCD system, a recombinational enzyme complex. In addition, the specialized physiology (aerobic and hyperthermophilic) of Aeropyrum spp. may also contribute to the entire-genome similarity. Despite having stable genomes, interference of synteny occurred with two proviruses, A. pernix spindle-shaped virus 1 (APSV1) and A. pernix ovoid virus 1 (APOV1), and clustered regularly interspaced short palindromic repeat (CRISPR) elements. Spacer sequences derived from the A. camini CRISPR showed significant matches with protospacers of the two proviruses infecting A. pernix, indicating that A. camini interacted with viruses closely related to APSV1 and APOV1. Furthermore, a significant fraction of the nonorthologous genes (41 to 45%) were proviral genes or ORFans probably originating from viruses. Although the genomes of A. camini and A. pernix were conserved, we observed nonsynteny that was attributed primarily to virus-related elements. Our findings indicated that the genomic diversification of Aeropyrum spp. is substantially caused by viruses.     

Angiotensin II induces the production of MMP-3 and MMP-13 through the MAPK signaling pathways via the AT1 receptor in osteoblasts

Angiotensin II (Ang II) plays an important role in the maintenance of bone mass and integrity by activation of the mitogen-activated protein kinases (MAPKs) and by modulation of balance between resorption by osteoclasts and formation by osteoblasts. However, the role of Ang II in the turnover of extracellular matrix (ECM) in osteoid by osteoblasts remains unclear. Therefore, we examined the effect of Ang II on the expression of matrix metalloproteinases (MMPs), plasminogen activators (PAs), and their inhibitors [i.e., tissue inhibitors of metalloproteinases (TIMPs) and PA inhibitor-1 (PAI-1)] using osteoblastic ROS17/2.8 cells. Treatment with Ang II strikingly increased the expressions of MMP-3 and -13 and promoted cell proliferation associated with reduced alkaline phosphatase activity as well as enhanced phosphorylated expression of extracellular signal-regulated kinase (ERK)1/2, p38 MAPK, and stress-activated protein kinases/c-jun N-terminal kinases (SAPK/JNK) in ROS17/2.8 cells. However, Ang II had no effect on the expression of MMP-2, -9, -14, urokinase-type PA, tissue-type PA, TIMP-1, -2, -3, and PAI-1 in cells. Losartan (AT₁ receptor blocker) blocked Ang II-induced expression of MMP-3 and -13, whereas PD123319 (AT₂ receptor blocker) did not completely block these responses. Losartan also blocked the Ang II-induced phosphorylation of ERK1/2, p38 MAPK, and SAPK/JNK. MAPK kinase 1/2 inhibitor PD98059 and JNK inhibitor SP600125 suppressed Ang II-induced expression of MMP-3 and -13. These results suggested that Ang II stimulated the degradation process that occurs during ECM turnover in osteoid by increasing the production of MMP-3 and -13 through MAPK signaling pathways via the AT₁ receptor in osteoblasts. Furthermore, our findings suggest that Ang II does not influence the plasminogen/plasmin pathway in osteoblasts.     

Replication‐coupled passive DNA demethylation for the erasure of genome imprints in mice

Genome‐wide DNA demethylation, including the erasure of genome imprints, in primordial germ cells (PGCs) is a critical first step to creating a totipotent epigenome in the germ line. We show here that, contrary to the prevailing model emphasizing active DNA demethylation, imprint erasure in mouse PGCs occurs in a manner largely consistent with replication‐coupled passive DNA demethylation: PGCs erase imprints during their rapid cycling with little de novo or maintenance DNA methylation potential and no apparent major chromatin alterations. Our findings necessitate the re‐evaluation of and provide novel insights into the mechanism of genome‐wide DNA demethylation in PGCs.     

Development and characterization of a novel set of microsatellite markers for Arisaema serratum (Araceae)

? Premise of the study: We developed novel microsatellite markers in Arisaema serratum, a perennial herb that possesses pitfall flowers and exhibits labile sex expression, to facilitate research on parentage and pollination biology in this species. ? Methods and Results: By using procedures for enrichment of desired microsatellite-containing fragments and PCR-based isolation of microsatellite arrays, we detected 18 novel microsatellite loci. Thirteen were highly polymorphic: the number of alleles per locus ranged from six to 46, the observed heterozygosities ranged from 0.320 to 0.940, and the expected heterozygosities ranged from 0.440 to 0.976. Nine of the 13 markers successfully amplified regions in congeneric species. ? Conclusions: These highly polymorphic markers will facilitate further studies on the mode of pollination and other aspects of reproductive biology in A. serratum.     

Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells

Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells.