Biomedical subjects

Tsuneyoshi Kuroiwa

Publications and source records attributed to Tsuneyoshi Kuroiwa.

At least 37 records · Page 2Linked to original sources

PubMed2003

Glom is a novel mitochondrial DNA packaging protein in Physarum polycephalum and causes intense chromatin condensation without suppressing DNA functions.

Mitochondrial DNA (mtDNA) is packed into highly organized structures called mitochondrial nucleoids (mt-nucleoids). To understand the organization of mtDNA and the overall regulation of its genetic activity within the mt-nucleoids, we identified and characterized a novel mtDNA packaging protein, termed Glom (a protein inducing agglomeration of mitochondrial chromosome), from highly condensed mt-nucleoids of the true slime mold, Physarum polycephalum. This protein could bind to the entire mtDNA and package mtDNA into a highly condensed state in vitro. Immunostaining analysis showed that Glom specifically localized throughout the mt-nucleoid. Deduced amino acid sequence revealed that Glom has a lysine-rich region with proline-rich domain in the N-terminal half and two HMG boxes in C-terminal half. Deletion analysis of Glom revealed that the lysine-rich region was sufficient for the intense mtDNA condensation in vitro. When the recombinant Glom proteins containing the lysine-rich region were expressed in Escherichia coli, the condensed nucleoid structures were observed in E. coli. Such in vivo condensation did not interfere with transcription or replication of E. coli chromosome and the proline-rich domain was essential to keep those genetic activities. The expression of Glom also complemented the E. coli mutant lacking the bacterial histone-like protein HU and the HMG-boxes region of Glom was important for the complementation. Our results suggest that Glom is a new mitochondrial histone-like protein having a property to cause intense DNA condensation without suppressing DNA functions.

Amino Acid Sequence↗

PubMed2003

Identification of higher plant GlsA, a putative morphogenesis factor of gametic cells.

GlsA has been identified in an asexual-reproductive-cell (gonidia)-deficient mutant of Volvox as a chaperone-like protein essential for gonidia production. In this study, we isolated an angiosperm glsA (LlglsA) gene expressed during Lilium longiflorum pollen development. Immunoblot analyses showed that the strong LlGlsA expression occurred in the generative cell and its pattern during pollen development corresponded to that of alpha-tubulin. Morphological analyses succeeded in visualizing the dispersion of the strong LlGlsA signal in developing generative cells. In addition, multiple-immunofluorescence staining of LlGlsA and alpha-tubulin revealed that some of the dot-like LlGlsA signals were co-localized with microtubule filaments. From those results, we suggest that angiosperm GlsA functions as a chaperone modifying various structures during male gametic cell formation.

Algal Proteins↗

PubMed2003

Complete sequence and analysis of the plastid genome of the unicellular red alga Cyanidioschyzon merolae.

The complete nucleotide sequence of the plastid genome of the unicellular primitive red alga Cyanidioschyzon merolae 10D (Cyanidiophyceae) was determined. The genome is a circular DNA composed of 149,987 bp with no inverted repeats. The G + C content of this plastid genome is 37.6%. The C. merolae plastid genome contains 243 genes, which are distributed on both strands and consist of 36 RNA genes (3 rRNAs, 31 tRNAs, tmRNA, and a ribonuclease P RNA component) and 207 protein genes, including unidentified open reading frames. The striking feature of this genome is the high degree of gene compaction; it has very short intergenic distances (approximately 40% of the protein genes were overlapped) and no genes have introns. This genome encodes several genes that are rarely found in other plastid genomes. A gene encoding a subunit of sulfate transporter (cysW) is the first to be identified in a plastid genome. The cysT and cysW genes are located in the C. merolae plastid genome in series, and they probably function together with other nuclear-encoded components of the sulfate transport system. Our phylogenetic results suggest that the Cyanidiophyceae, including C. merolae, are a basal clade within the red lineage plastids.

Amino Acid Sequence↗

PubMed2003

Dynamic recruitment of dynamin for final mitochondrial severance in a primitive red alga.

Dynamins are a eukaryote-specific family of GTPases. Some family members are involved in diverse and varied cellular activities. Here, we report that the primitive red alga Cyanidioschyzon merolae retains only one dynamin homolog, CmDnm1, belonging to the mitochondrial division subfamily. Previously, the bacterial cell division protein, FtsZ, was shown to localize at the mitochondrial division site in the alga. We showed that FtsZ and dynamin coexist as mitochondrial division-associated proteins that act during different phases of division. CmDnm1 was recruited from 10-20 cytoplasmic patches (dynamin patches) to the midpoint of the constricted mitochondrion-dividing ring (MD ring), which was observed as an electron-dense structure on the cytoplasmic side. CmDnm1 is probably not required for early constriction; it forms a ring or spiral when the outer mitochondrial membrane is finally severed, whereas the FtsZ and MD rings are formed before constriction. It is thought that the FtsZ, MD, and dynamin rings are involved in scaffolding, constriction, and final separation, respectively. In eukaryotes, mitochondrial severance is probably the most conserved role for the dynamin family.

Blotting, Western↗

PubMed2003

The phylogenetic position of red algae revealed by multiple nuclear genes from mitochondria-containing eukaryotes and an alternative hypothesis on the origin of plastids.

Red algae are one of the main photosynthetic eukaryotic lineages and are characterized by primitive features, such as a lack of flagella and the presence of phycobiliproteins in the chloroplast. Recent molecular phylogenetic studies using nuclear gene sequences suggest two conflicting hypotheses (monophyly versus non-monophyly) regarding the relationships between red algae and green plants. Although kingdom-level phylogenetic analyses using multiple nuclear genes from a wide-range of eukaryotic lineages were very recently carried out, they used highly divergent gene sequences of the cryptomonad nucleomorph (as the red algal taxon) or incomplete red algal gene sequences. In addition, previous eukaryotic phylogenies based on nuclear genes generally included very distant archaebacterial sequences (designated as the outgroup) and/or amitochondrial organisms, which may carry unusual gene substitutions due to parasitism or the absence of mitochondria. Here, we carried out phylogenetic analyses of various lineages of mitochondria-containing eukaryotic organisms using nuclear multigene sequences, including the complete sequences from the primitive red alga Cyanidioschyzon merolae. Amino acid sequence data for two concatenated paralogous genes (alpha- and beta-tubulin) from mitochondria-containing organisms robustly resolved the basal position of the cellular slime molds, which were designated as the outgroup in our phylogenetic analyses. Phylogenetic analyses of 53 operational taxonomic units (OTUs) based on a 1525-amino-acid sequence of four concatenated nuclear genes (actin, elongation factor-1alpha, alpha-tubulin, and beta-tubulin) reliably resolved the phylogeny only in the maximum parsimonious (MP) analysis, which indicated the presence of two large robust monophyletic groups (Groups A and B) and the basal eukaryotic lineages (red algae, true slime molds, and amoebae). Group A corresponded to the Opisthokonta (Metazoa and Fungi), whereas Group B included various primary and secondary plastid-containing lineages (green plants, glaucophytes, euglenoids, heterokonts, and apicomplexans), Ciliophora, Kinetoplastida, and Heterolobosea. The red algae represented the sister lineage to Group B. Using 34 OTUs for which essentially the entire amino acid sequences of the four genes are known, MP, distance, quartet puzzling, and two types of maximum likelihood (ML) calculations all robustly resolved the monophyly of Group B, as well as the basal position of red algae within eukaryotic organisms. In addition, phylogenetic analyses of a concatenated 4639-amino-acid sequence for 12 nuclear genes (excluding the EF-2 gene) of 12 mitochondria-containing OTUs (including C. merolae) resolved a robust non-sister relationship between green plants and red algae within a robust monophyletic group composed of red algae and the eukaryotic organisms belonging to Group B. A new scenario for the origin and evolution of plastids is suggested, based on the basal phylogenetic position of the red algae within the large clade (Group B plus red algae). The primary plastid endosymbiosis likely occurred once in the common ancestor of this large clade, and the primary plastids were subsequently lost in the ancestor(s) of the Discicristata (euglenoids, Kinetoplastida, and Heterolobosea), Heterokontophyta, and Alveolata (apicomplexans and Ciliophora). In addition, a new concept of "Plantae" is proposed for phototrophic and nonphototrophic organisms belonging to Group B and red algae, on the basis of the common history of the primary plastid endosymbiosis. The Plantae include primary plastid-containing phototrophs and nonphototrophic eukaryotes that possibly contain genes of cyanobacterial origin acquired in the primary endosymbiosis.

Cell Nucleus↗

PubMed2003

Phylogeny of plastids based on cladistic analysis of gene loss inferred from complete plastid genome sequences.

Based on the recent hypothesis on the origin of eukaryotic phototrophs, red algae, green plants, and glaucophytes constitute the "primary photosynthetic eukaryotes" (whose plastids may have originated directly from a cyanobacterium-like prokaryote via primary endosymbiosis), whereas the plastids of other lineages of eukaryotic phototrophs appear to be the result of secondary or tertiary endosymbiotic events (involving a phototrophic eukaryote and a host cell). Although phylogenetic analyses using multiple plastid genes from a wide range of eukaryotic lineages have been carried out, some of the major phylogenetic relationships of plastids remain ambiguous or conflict between different phylogenetic methods used for nucleotide or amino acid substitutions. Therefore, an alternative methodology to infer the plastid phylogeny is needed. Here, we carried out a cladistic analysis of the "loss of plastid genes" after primary endosymbiosis using complete plastid genome sequences from a wide range of eukaryotic phototrophs. Since it is extremely unlikely that plastid genes are regained during plastid evolution, we used the irreversible Camin-Sokal model for our cladistic analysis of the loss of plastid genes. The cladistic analysis of the 274 plastid protein-coding genes resolved the 20 operational taxonomic units representing a wide range of eukaryotic lineages (including three secondary plastid-containing groups) into two large monophyletic groups with high bootstrap values: one corresponded to the red lineage and the other consisted of a large clade composed of the green lineage (green plants and Euglena) and the basal glaucophyte plastid. Although the sister relationship between the green lineage and the Glaucophyta was not resolved in recent phylogenetic studies using amino acid substitutions from multiple plastid genes, it is consistent with the rbcL gene phylogeny and with a recent phylogenetic study using multiple nuclear genes. In addition, our analysis robustly resolved the conflicting/ambiguous phylogenetic positions of secondary plastids in previous phylogenetic studies: the Euglena plastid was sister to the chlorophycean (Chlamydomonas) lineage, and the secondary plastids from the diatom (Odontiella) and cryptophyte (Guillardia) were monophyletic within the red lineage.

Animals↗

PubMed2003

Phylogeny of the quadriflagellate Volvocales (Chlorophyceae) based on chloroplast multigene sequences.

Since the phylogenetic relationships of the green plants (green algae and land plants) have been extensively studied using 18S ribosomal RNA sequences, change in the arrangement of basal bodies in flagellate cells is considered to be one of the major evolutionary events in the green plants. However, the phylogenetic relationships between biflagellate and quadriflagellate species within the Volvocales remain uncertain. This study examined the phylogeny of three genera of quadriflagellate Volvocales (Carteria, Pseudocarteria, and Hafniomonas) using concatenated sequences from three chloroplast genes. Using these multigene sequences, all three quadriflagellate genera were basal to other members (biflagellates) of the CW (clockwise) group (the Volvocales and their relatives, the Chlorophyceae) and formed three robust clades. Since the flagellar apparatuses of these three quadriflagellate lineages are diverse, including counter clockwise (CCW) and CW orientation of the basal bodies, the CW orientation of the basal bodies might have evolved from the CCW orientation in the ancestral quadriflagellate volvocalean algae, giving rise to the biflagellates, major members of the CW group.

Chlorophyta↗

PubMed2003

An evolutionary puzzle: chloroplast and mitochondrial division rings.

Consistent with their bacterial origin, chloroplasts and primitive mitochondria retain a FtsZ ring for division. However, chloroplasts and mitochondria have lost most of the proteins required for bacterial division other than FtsZ and certain homologues of the Min proteins, but they do contain plastid and mitochondrion dividing rings, which were recently shown to be distinct from the FtsZ ring. Moreover, recent studies have revealed that rings of the eukaryote-specific dynamin-related family of GTPases regulate the division of chloroplasts and mitochondria, and these proteins emerged early in eukaryotic evolution. These findings suggest that the division of chloroplasts and primitive mitochondria involve very similar systems, consisting of an amalgamation of rings from bacteria and eukaryotes.

Animals↗

PubMed2003

Pollen-tube guidance: beacons from the female gametophyte.

The sperm cell of a flowering plant cannot migrate unaided and it must be transported by the pollen-tube cell before successful fertilization can occur. The pollen tube is precisely guided to the target female gametophyte, the embryo sac, which contains the egg cell. The mechanism that precisely directs the pollen tube through the pistil to the female gametophyte has been studied for more than a century. There has been controversy over whether a diffusible signal attracts the pollen tube or whether female tissues define its path. Emerging genetic and physiological data show that the female gametophyte produces at least two directional signals, and that at least one of these signals is diffusible and derived from the two synergid cells.

Arabidopsis↗

PubMed2003

Activation of cell proliferation by brassinolide application in tobacco BY-2 cells: effects of brassinolide on cell multiplication, cell-cycle-related gene expression, and organellar DNA contents.

Brassinosteroids (BRs) are steroidal phytohormones that are essential for many processes in plant growth and development, such as cell expansion, vascular differentiation, and responses to stress. The effects of BRs on cell division are unclear, as attested by contradictory published results. To determine the effect of BRs on cell division, the tobacco (Nicotiana tabacum) BY-2 cell line, which is a widely-used model system in plant cell biology, was used. It was found that brassinolide (BL) promoted cell division only during the early phase of culture and in the absence of auxin (2,4-D). This promotion of cell division was confirmed by RNA gel blot analyses using cell-cycle-related gene probes. At later stages in the culturing periods of BL-supplied and 2,4-D-supplied BY-2 cells, differences in cell multiplication and cell-cycle-related gene expression were observed. Moreover, the BL-treated BY-2 cells had morphological differences from the 2,4-D-treated cells. To determine whether suppressed organellar DNA replication limited this promotion of cell division during the early culture phase, this replication was examined and it was found that BL treatment had no effect on activating organellar (plastid- and mitochondrial-) DNA synthesis. As preferential organellar DNA synthesis, which is activated by 2,4-D, is necessary during successive cell divisions in BY-2 cells, these data suggest that the mechanism of the promotion of cell division by BL treatment is distinct from that regulated by the balance of auxin and cytokinin.

Brassinosteroids↗

PubMed2003

A plant-specific dynamin-related protein forms a ring at the chloroplast division site.

Chloroplasts have retained the bacterial FtsZ for division, whereas mitochondria lack FtsZ except in some lower eukaryotes. Instead, mitochondrial division involves a dynamin-related protein, suggesting that chloroplasts retained the bacterial division system, whereas a dynamin-based system replaced the bacterial system in mitochondria during evolution. In this study, we identified a novel plant-specific group of dynamins from the primitive red alga Cyanidioschyzon merolae. Synchronization of chloroplast division and immunoblot analyses showed that the protein (CmDnm2) associates with the chloroplast only during division. Immunocytochemical analyses showed that CmDnm2 appears in cytoplasmic patches just before chloroplast division and is recruited to the cytosolic side of the chloroplast division site to form a ring in the late stage of division. The ring constricts until division is complete, after which it disappears. These results show that a dynamin-related protein also participates in chloroplast division and that its behavior differs from that of FtsZ and plastid-dividing rings that form before constriction at the site of division. Combined with the results of a recent study of mitochondrial division in Cyanidioschyzon, our findings led us to hypothesize that when first established in lower eukaryotes, mitochondria and chloroplasts divided using a very similar system that included the FtsZ ring, the plastid-dividing/mitochondrion-dividing ring, and the dynamin ring.

Algal Proteins↗

PubMed2002

Reduction in amounts of mitochondrial DNA in the sperm cells as a mechanism for maternal inheritance in Hordeum vulgare.

It is known that extranuclear organelle DNA is inherited maternally in the majority of angiosperms. The mechanisms for maternal inheritance have been well studied in plastids but not in mitochondria. In the present study we examined the mitochondrial DNA in the male reproductive cells of Hordeum vulgare L. by immunoelectron microscopy. Our results show that the number of anti-DNA gold particles on sections of sperm cell mitochondria decreased by 97% during pollen development. The reduction occurred rapidly in the generative cells and subsequently in the sperm cells, concomitant with a remarkable reduction in mitochondrial volume. It seems that the copy numbers of mitochondrial DNA were reduced in the male reproductive cells, which may be a possible mechanism by which paternal transmission is inhibited. Unlike mitochondria, plastids are excluded from the generative cells during the first pollen mitosis. These data suggest a mechanism for maternal inheritance of mitochondria in angiosperms and for independent control of inheritance of mitochondria and plastids in H. vulgare.

DNA, Mitochondrial↗

PubMed2002

Characterization of a putative fusogen encoded in a mitochondrial plasmid of Physarum polycephalum.

The mitochondrial plasmid mF induces mitochondrial fusion in zygotes and during sporulation in the true slime mold Physarum polycephalum. There are nine open reading frames (ORFs) in the mF plasmid, and it has been suggested that ORF640 encodes the mitochondrial fusogen. We prepared antisera directed against the ORF640 protein (ORF640p) in rabbits, and used it to localize this protein in mitochondria. Western blot analysis showed that ORF640p was produced only in the mitochondria of mF(+) strains, i.e., in cells that carried the mF plasmid. Proteinase K treatment of mitochondria isolated from the mF(+) strain suggested that the C-terminus coiled-coil (CC) region of ORF640p was exported from the matrix to the cytosol. Digitonin treatment confirmed this localization. West-Western blot analysis suggested that the CC region of ORF640p formed multimers and could interfere with an unknown mitochondrial protein in normal mitochondria. These results suggest that ORF640p is related to fusion of the outer mitochondrial membrane.

Journal Article↗

PubMed2002

An mt(+) gamete-specific nuclease that targets mt(-) chloroplasts during sexual reproduction in C. reinhardtii.

Although the active digestion of mating-type minus (mt-) chloroplast DNA (cpDNA) in young zygotes is considered to be the basis for the uniparental inheritance of cpDNA in Chlamydomonas reinhardtii, little is known about the underlying molecular mechanism. One model of active digestion proposes that nucleases are either synthesized or activated to digest mt- cpDNA. We used a native-PAGE/in gelo assay to investigate nuclease activities in chloroplasts from young zygotes, and identified a novel Ca(2+)-dependent nuclease activity. The timing of activation (approximately 60-90 min after mating) and the localization of the nuclease activity (in mt- chloroplasts) coincided with the active digestion of mt- cpDNA. Furthermore, the activity of the nuclease was coregulated with the maturation of mating-type plus (mt+) gametes, which would enable the efficient digestion of mt- cpDNA. Based on these observations, we propose that the nuclease (designated as Mt(+)-specific DNase, MDN) is a developmentally controlled nuclease that is activated in mt+ gametes and participates in the destruction of mt- cpDNA in young zygotes, thereby ensuring uniparental inheritance of chloroplast traits.

Animals↗

PubMed2002

Chloroplast division machinery as revealed by immunofluorescence and electron microscopy.

The division of chloroplasts (plastids) is critical for the viability of photosynthetic eukaryotes. Previously we reported on the chloroplast division apparatus, which consists of inner and outer double or triple rings (PD rings). Chloroplasts are assumed to arise from bacterial endosymbionts, while bacterial division is instigated by a bacterial cytokinesis Z-ring protein (FtsZ). Here we present immunofluorescence and electron-microscopic evidence of chloroplast division via complex machinery involving the FtsZ and PD rings in the higher plant Pelargonium zonale Ait. Prior to invagination, the FtsZ protein was attached to a ring at the stromal division site. Following formation of the FtsZ ring, the inner stromal and outer cytosolic PD rings appeared, signifying the initiation of invagination. The FtsZ ring and the PD rings were found at the leading edge of chloroplast constriction throughout division. During chloroplast division, neither the FtsZ nor the inner rings changed width, but the volume of the outer ring gradually increased. We suggest that the FtsZ ring determines the division region, after which the inner and outer PD rings are formed as a lining for the FtsZ ring. With the outer ring providing the motivating force, the FtsZ and inner PD rings ultimately decompose to their base components.

Antibodies↗

PubMed2002

Three-dimensional progression of programmed death in the rice coleoptile.

Plant death during development is a highly orchestrated process at the cellular, tissue, organ, and whole-plant levels. The process toward death is endogenously programmed in plants. With our original approach called "three-dimensional analysis" using the rice coleoptile, we revealed detailed morphological alterations in the progression of senescence and programmed cell death involved in the air space (aerenchyma) formation at both tissue and cellular levels. Although these two types of cell death exhibited a distinct pattern of progression at the tissue level, the set of intracellular events was highly conserved. From those comprehensive investigations, we hypothesized that the identical program of death functions in each process of cell death, and that the initiation and progression of cell death is highly regulated by the environmental input.

Apoptosis↗

PubMed2002

A complex and mobile structure forms a distinct subregion within the continuous vacuolar membrane in young cotyledons of Arabidopsis.

The plant vacuole is a multifunctional organelle which is essential for growth and development. To visualize the dynamics of plant vacuolar membranes, gamma-TIP (tonoplast intrinsic protein) was fused to GFP and expressed in Arabidopsis thaliana. The marker molecule was targeted to the vacuolar membranes in most tissues, as expected. In rapidly expanding cells, some additional spherical structures were often observed within the lumen of vacuoles, which emitted strong fluorescence. To confirm their normal presence, we examined wild-type Arabidopsis cotyledons by transmission electron microscopy. The metal-contact rapid-freezing method revealed that the vacuolar lumen of epidermal cells contained many cytoplasmic projections, which often formed spherical structures (1-3 microm diameter) consisting of double membranes. Thus we concluded that these structures are authentic and named them 'bulbs'. Three-dimensional reconstruction from serial electron microscopic images demonstrates that bulbs are very intricately folded, but are continuous with the limiting vacuolar membrane. The fluorescence intensity of bulbs is about threefold higher than that of vacuolar membrane. GFP-AtRab75c, another marker of the vacuole, did not give fluorescent signals of bulbs in transgenic plants, but the existence of bulbs was still confirmed by electron microscopy. These results suggest that bulbs define a subregion in the continuous vacuolar membrane, where some proteins are concentrated and others segregated.

Arabidopsis↗

PubMed2002

Isolation and expression of a novel starch-storing cell-specific gene containing the KH RNA binding domain from tobacco-cultured cells BY-2.

In cultured Bright Yellow-2 tobacco (Nicotiana tabacum) cells, the depletion of 2,4-dichlorophenoxyacetic acid (2,4-D) in the culture medium induces amyloplast development. This differentiation also includes a decrease in cell multiplication, and an increase in cell size. These changes were primarily triggered by the depletion of 2,4-D, and accelerated by the addition of benzyladenine (BA). Three cDNAs were identified whose transcript levels are specifically increased during differentiation of starch-storing cells using the differential display method, and designated as starch-storing cell induced genes (SCI genes). One of these cDNAs, SCI2 encodes a 285 amino acids long protein with a KH RNA-binding domain. A database search revealed that this protein showed similarity to respective domains of mammalian quaking proteins. 2,4-D addition, which can convert starch-storing cells into dividing cells, to starch-storing BY-2 cells, immediately decreases the SCI2 transcript level, suggesting that SCI2 may have some role in starch-storing cell differentiation in BY-2 cells.

2,4-Dichlorophenoxyacetic Acid↗