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Kan Tanaka - One of the best experts on this subject based on the ideXlab platform.
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overexpression of a glycogenin cmglg2 enhances floridean starch accumulation in the red alga Cyanidioschyzon Merolae
Plant Signaling & Behavior, 2019Co-Authors: Imran Pancha, Kan Tanaka, Sousuke ImamuraAbstract:Microalgae accumulate energy-reserved molecules, such as triacylglycerol and carbohydrates, which are suitable feedstocks for renewable energies such as biodiesel and bioethanol. However, the molecular mechanisms behind the microalgae accumulating these molecules require further elucidation. Recently, we have reported that the target of rapamycin (TOR)-signaling is a major pathway to regulate floridean starch synthesis by changing the phosphorylation status of CmGLG1, a glycogenin generally required for the initiation of starch/glycogen synthesis, in the unicellular red alga Cyanidioschyzon Merolae. In the present study, we confirmed that another glycogenin, CmGLG2, is also involved in the floridean starch synthesis in this alga, since the CmGLG2 overexpression resulted in a two-fold higher floridean starch content in the cell. The results indicate that both glycogenin isoforms play an important role in floridean starch synthesis in C. Merolae, and would be a potential target for improvement of floridean starch production in microalgae.
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target of rapamycin signaling modulates starch accumulation via glycogenin phosphorylation status in the unicellular red alga Cyanidioschyzon Merolae
Plant Journal, 2019Co-Authors: Imran Pancha, Kan Tanaka, Hiroki Shima, Nahoko Higashitani, Kazuhiko Igarashi, Atsushi Higashitani, Sousuke ImamuraAbstract:The target of rapamycin (TOR) signaling pathway is involved in starch accumulation in various eukaryotic organisms; however, the molecular mechanism behind this phenomenon in eukaryotes has not been elucidated. We report a regulatory mechanism of starch accumulation by TOR in the unicellular red alga, Cyanidioschyzon Merolae. The starch content in C. Merolae after TOR-inactivation by rapamycin, a TOR-specific inhibitor, was increased by approximately 10-fold in comparison with its drug vehicle, dimethyl sulfoxide. However, our previous transcriptome analysis showed that the expression level of genes related to carbohydrate metabolism was unaffected by rapamycin, indicating that starch accumulation is regulated at post-transcriptional levels. In this study, we performed a phosphoproteome analysis using liquid chromatography-tandem mass spectrometry to investigate potential post-transcriptional modifications, and identified 52 proteins as candidate TOR substrates. Among the possible substrates, we focused on the function of CmGLG1, because its phosphorylation at the Ser613 residue was decreased after rapamycin treatment, and overexpression of CmGLG1 resulted in a 4.7-fold higher starch content. CmGLG1 is similar to the priming protein, glycogenin, which is required for the initiation of starch/glycogen synthesis, and a budding yeast complementation assay demonstrated that CmGLG1 can functionally substitute for glycogenin. We found an approximately 60% reduction in the starch content in a phospho-mimicking CmGLG1 overexpression strain, in which Ser613 was substituted with aspartic acid, in comparison with the wild-type CmGLG1 overexpression cells. Our results indicate that TOR modulates starch accumulation by changing the phosphorylation status of the CmGLG1 Ser613 residue in C. Merolae.
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a myb type transcription factor myb2 represses light harvesting protein genes in Cyanidioschyzon Merolae
FEBS Letters, 2017Co-Authors: Yasuko Kawase, Sousuke Imamura, Kan TanakaAbstract:While searching for transcriptional regulators that respond to changes in light regimes, we identified a MYB domain-containing protein, MYB2, that accumulates under dark and other conditions in the unicellular red alga Cyanidioschyzon Merolae. The isolation and analysis of a MYB2 mutant revealed that MYB2 represses the expression of the nuclear-encoded chloroplast RNA polymerase sigma factor gene SIG2, which results in the repression of the chloroplast-encoded phycobilisome genes that are under its control. Since nuclear-encoded phycobilisome and other light-harvesting protein genes are also repressed by MYB2, we conclude that MYB2 has a role in repressing the expression of light-harvesting genes. The MYB2 mutant is sensitive to a prolonged dark incubation, indicating the importance of MYB2 for cell viability in the dark.
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Transcriptional regulation of tetrapyrrole biosynthetic genes explains abscisic acid-induced heme accumulation in the unicellular red alga Cyanidioschyzon Merolae
Frontiers Media S.A., 2016Co-Authors: Yuki Kobayashi, Kan TanakaAbstract:Abscisic acid (ABA), a pivotal phytohormone that is synthesized in response to abiotic stresses and other environmental changes, induces various physiological responses. Heme, in its unbound form, has a positive signaling role in cell-cycle initiation in Cyanidioschyzon Merolae. ABA induces heme accumulation, but also prevents cell-cycle initiation through the titration of the unbound heme by inducing the heme scavenging protein tryptophan-rich sensory protein-related protein O. In this study, we analyzed the accumulation of tetrapyrrole biosynthetic gene transcripts after the addition of ABA to the medium and found that transcripts of a ferrochelatase and a magnesium-chelatase subunit increased, while other examined transcripts decreased. Under the same conditions, the heme and magnesium-protoporphyrin IX contents increased, while the protoporphyrin IX content decreased. Thus, ABA may regulate the intracellular heme and other tetrapyrrole contents through the transcriptional regulation of biosynthetic genes
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nuclear encoded chloroplast rna polymerase sigma factor sig2 activates chloroplast encoded phycobilisome genes in a red alga Cyanidioschyzon Merolae
FEBS Letters, 2013Co-Authors: Mitsumasa Hanaoka, Sousuke Imamura, Gaku Fujii, Kan TanakaAbstract:The phycobilisome (PBS) is a photosynthetic light-harvesting complex in red algae, whose structural genes are separately encoded by both the nuclear and chloroplast genomes. While the expression of PBS genes in both genomes is responsive to environmental changes to modulate light-harvesting efficiency, little is known about how gene expression of the two genomes is coordinated. In this study, we focused on the four nuclear-encoded chloroplast sigma factors to understand aspects of this coordination, and found that SIG2 directs the expression of chloroplast PBS genes in the red alga Cyanidioschyzon Merolae.
Tsuneyoshi Kuroiwa - One of the best experts on this subject based on the ideXlab platform.
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Evolutionary significance of the ring-like plastid nucleus in the primitive red alga Cyanidioschyzon Merolae as revealed by drying
Protoplasma, 2020Co-Authors: Tsuneyoshi Kuroiwa, Mio Ohnuma, Yuuta Imoto, Fumi Yagisawa, Osami Misumi, Noriko Nagata, Haruko KuroiwaAbstract:Primary plastids originated from a free-living cyanobacterial ancestor and possess their own genomes—probably a few DNA copies. These genomes, which are organized in centrally located plastid nuclei (CN-type pt-nuclei), are produced from preexisting plastids by binary division. Ancestral algae with a CN-type pt-nucleus diverged and evolved into two basal eukaryotic lineages: red algae with circular (CL-type) pt-nuclei and green algae with scattered small (SN-type) pt-nuclei. Although the molecular dynamics of pt-nuclei in green algae and plants are now being analyzed, the process of the conversion of the original algae with a CN-type pt-nucleus to red algae with a CL-type one has not been studied. Here, we show that the CN-type pt-nucleus in the primitive red alga Cyanidioschyzon Merolae can be changed to the CL-type by application of drying to produce slight cell swelling. This result implies that CN-type pt-nuclei are produced by compact packing of CL-type ones, which suggests that a C. Merolae– like alga was the original progenitor of the red algal lineage. We also observed that the CL-type pt-nucleus has a chain-linked bead-like structure. Each bead is most likely a small unit of DNA, similar to CL-type pt-nuclei in brown algae. Our results thus suggest a C. Merolae –like alga as the candidate for the secondary endosymbiont of brown algae.
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dynamics of the nucleoside diphosphate kinase protein dynamo2 correlates with the changes in the global gtp level during the cell cycle of Cyanidioschyzon Merolae
Proceedings of the Japan Academy. Ser. B: Physical and Biological Sciences, 2019Co-Authors: Tsuneyoshi Kuroiwa, Mio Ohnuma, Yuuta Imoto, Haruko Kuroiwa, Yuichi Abe, Kanji Okumoto, Yukio FujikiAbstract:GTP is an essential source of energy that supports a large array of cellular mechanochemical structures ranging from protein synthesis machinery to cytoskeletal apparatus for maintaining the cell cycle. However, GTP regulation during the cell cycle has been difficult to investigate because of heterogenous levels of GTP in asynchronous cell cycles and genetic redundancy of the GTP-generating enzymes. Here, in the unicellular red algae Cyanidioschyzon Merolae, we demonstrated that the ATP-GTP-converting enzyme DYNAMO2 is an essential regulator of global GTP levels during the cell cycle. The cell cycle of C. Merolae can be highly synchronized by light/dark stimulations to examine GTP levels at desired time points. Importantly, the genome of C. Merolae encodes only two isoforms of the ATP-GTP-converting enzyme, namely DYNAMO1 and DYNAMO2. DYNAMO1 regulates organelle divisions, whereas DYNAMO2 is entirely localized in the cytoplasm. DYNAMO2 protein levels increase during the S-M phases, and changes in GTP levels are correlated with these DYNAMO2 protein levels. These results indicate that DYNAMO2 is a potential regulator of global GTP levels during the cell cycle.
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algae sense exact temperatures small heat shock proteins are expressed at the survival threshold temperature in Cyanidioschyzon Merolae and chlamydomonas reinhardtii
Genome Biology and Evolution, 2014Co-Authors: Yusuke Kobayashi, Tsuneyoshi Kuroiwa, Takayuki Fujiwara, Naomi Harada, Yoshiki Nishimura, Takafumi Saito, Mami Nakamura, Osami MisumiAbstract:The primitive red alga Cyanidioschyzon Merolae inhabits acidic hot springs and shows robust resistance to heat shock treatments up to 63 °C. Microarray analysis was performed to identify the key genes underlying the high temperature tolerance of this organism. Among the upregulated genes that were identified, we focused on two small heat shock proteins (sHSPs) that belong to a unique class of HSP families. These two genes are located side by side in an inverted repeat orientation on the same chromosome and share a promoter. These two genes were simultaneously and rapidly upregulated in response to heat shock treatment (>1,000-fold more than the control). Interestingly, upregulation appeared to be triggered not by a difference in temperatures, but rather by the absolute temperature. Similar sHSP structural genes have been reported in the green alga Chlamydomonas reinhardtii, but the threshold temperature for the expression of these sHSP-encoding genes in Ch. reinhardtii was different from the threshold temperature for the expression of the sHSP genes from Cy. Merolae. These results indicate the possible importance of an absolute temperature sensing system in the evolution and tolerance of high-temperature conditions among unicellular microalgae.
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gene targeting in the red alga Cyanidioschyzon Merolae single and multi copy insertion using authentic and chimeric selection markers
PLOS ONE, 2013Co-Authors: Takayuki Fujiwara, Tsuneyoshi Kuroiwa, Mio Ohnuma, Masaki Yoshida, Tatsuya HiranoAbstract:The unicellular red alga Cyanidioschyzon Merolae is an emerging model organism for studying organelle division and inheritance: the cell is composed of an extremely simple set of organelles (one nucleus, one mitochondrion and one chloroplast), and their genomes are completely sequenced. Although a fruitful set of cytological and biochemical methods have now been developed, gene targeting techniques remain to be fully established in this organism. Thus far, only a single selection marker, URACm-Gs, has been available that complements the uracil-auxotrophic mutant M4. URACm-Gs, a chimeric URA5.3 gene of C. Merolae and the related alga Galdieria sulphuraria, was originally designed to avoid gene conversion of the mutated URA5.3 allele in the parental strain M4. Although an early example of targeted gene disruption by homologous recombination was reported using this marker, the genome structure of the resultant transformants had never been fully characterized. In the current study, we showed that the use of the chimeric URACm-Gs selection marker caused multicopy insertion at high frequencies, accompanied by undesired recombination events at the targeted loci. The copy number of the inserted fragments was variable among the transformants, resulting in high yet uneven levels of transgene expression. In striking contrast, when the authentic URA5.3 gene (URACm-Cm) was used as a selection marker, efficient single-copy insertion was observed at the targeted locus. Thus, we have successfully established a highly reliable and reproducible method for gene targeting in C. Merolae. Our method will be applicable to a number of genetic manipulations in this organism, including targeted gene disruption, replacement and tagging.
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mitotic inheritance of endoplasmic reticulum in the primitive red alga Cyanidioschyzon Merolae
Protoplasma, 2012Co-Authors: Yuuta Imoto, Fumi Yagisawa, Haruko Kuroiwa, Takayuki Fujiwara, Keiji Nishida, Tsuneyoshi KuroiwaAbstract:Endoplasmic reticulum (ER) is a major site for secretory protein folding and lipid synthesis. Since ER cannot be synthesized de novo, it must be inherited during the cell cycle. Studying ER inheritance can however be difficult because the ER of typical plant and animal cells is morphologically complex. Therefore, our study used Cyanidioschyzon Merolae, a species that has a simple ER structure, to investigate the inheritance of this organelle. Using immunofluorescence microscopy, we demonstrated that C. Merolae contains a nuclear ER (nuclear envelope) and a small amount of peripheral ER extending from the nuclear ER. During mitosis, the nuclear ER became dumbbell-shaped and underwent division. Peripheral ER formed ring-like structures during the G1 and S phases, and extended toward the mitochondria and cell division planes during the M phase. These observations indicated that C. Merolae undergoes closed mitosis, whereby the nuclear ER does not diffuse, and the peripheral ER contains cell cycle-specific structures.
Mio Ohnuma - One of the best experts on this subject based on the ideXlab platform.
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Evolutionary significance of the ring-like plastid nucleus in the primitive red alga Cyanidioschyzon Merolae as revealed by drying
Protoplasma, 2020Co-Authors: Tsuneyoshi Kuroiwa, Mio Ohnuma, Yuuta Imoto, Fumi Yagisawa, Osami Misumi, Noriko Nagata, Haruko KuroiwaAbstract:Primary plastids originated from a free-living cyanobacterial ancestor and possess their own genomes—probably a few DNA copies. These genomes, which are organized in centrally located plastid nuclei (CN-type pt-nuclei), are produced from preexisting plastids by binary division. Ancestral algae with a CN-type pt-nucleus diverged and evolved into two basal eukaryotic lineages: red algae with circular (CL-type) pt-nuclei and green algae with scattered small (SN-type) pt-nuclei. Although the molecular dynamics of pt-nuclei in green algae and plants are now being analyzed, the process of the conversion of the original algae with a CN-type pt-nucleus to red algae with a CL-type one has not been studied. Here, we show that the CN-type pt-nucleus in the primitive red alga Cyanidioschyzon Merolae can be changed to the CL-type by application of drying to produce slight cell swelling. This result implies that CN-type pt-nuclei are produced by compact packing of CL-type ones, which suggests that a C. Merolae– like alga was the original progenitor of the red algal lineage. We also observed that the CL-type pt-nucleus has a chain-linked bead-like structure. Each bead is most likely a small unit of DNA, similar to CL-type pt-nuclei in brown algae. Our results thus suggest a C. Merolae –like alga as the candidate for the secondary endosymbiont of brown algae.
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dynamics of the nucleoside diphosphate kinase protein dynamo2 correlates with the changes in the global gtp level during the cell cycle of Cyanidioschyzon Merolae
Proceedings of the Japan Academy. Ser. B: Physical and Biological Sciences, 2019Co-Authors: Tsuneyoshi Kuroiwa, Mio Ohnuma, Yuuta Imoto, Haruko Kuroiwa, Yuichi Abe, Kanji Okumoto, Yukio FujikiAbstract:GTP is an essential source of energy that supports a large array of cellular mechanochemical structures ranging from protein synthesis machinery to cytoskeletal apparatus for maintaining the cell cycle. However, GTP regulation during the cell cycle has been difficult to investigate because of heterogenous levels of GTP in asynchronous cell cycles and genetic redundancy of the GTP-generating enzymes. Here, in the unicellular red algae Cyanidioschyzon Merolae, we demonstrated that the ATP-GTP-converting enzyme DYNAMO2 is an essential regulator of global GTP levels during the cell cycle. The cell cycle of C. Merolae can be highly synchronized by light/dark stimulations to examine GTP levels at desired time points. Importantly, the genome of C. Merolae encodes only two isoforms of the ATP-GTP-converting enzyme, namely DYNAMO1 and DYNAMO2. DYNAMO1 regulates organelle divisions, whereas DYNAMO2 is entirely localized in the cytoplasm. DYNAMO2 protein levels increase during the S-M phases, and changes in GTP levels are correlated with these DYNAMO2 protein levels. These results indicate that DYNAMO2 is a potential regulator of global GTP levels during the cell cycle.
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gene targeting in the red alga Cyanidioschyzon Merolae single and multi copy insertion using authentic and chimeric selection markers
PLOS ONE, 2013Co-Authors: Takayuki Fujiwara, Tsuneyoshi Kuroiwa, Mio Ohnuma, Masaki Yoshida, Tatsuya HiranoAbstract:The unicellular red alga Cyanidioschyzon Merolae is an emerging model organism for studying organelle division and inheritance: the cell is composed of an extremely simple set of organelles (one nucleus, one mitochondrion and one chloroplast), and their genomes are completely sequenced. Although a fruitful set of cytological and biochemical methods have now been developed, gene targeting techniques remain to be fully established in this organism. Thus far, only a single selection marker, URACm-Gs, has been available that complements the uracil-auxotrophic mutant M4. URACm-Gs, a chimeric URA5.3 gene of C. Merolae and the related alga Galdieria sulphuraria, was originally designed to avoid gene conversion of the mutated URA5.3 allele in the parental strain M4. Although an early example of targeted gene disruption by homologous recombination was reported using this marker, the genome structure of the resultant transformants had never been fully characterized. In the current study, we showed that the use of the chimeric URACm-Gs selection marker caused multicopy insertion at high frequencies, accompanied by undesired recombination events at the targeted loci. The copy number of the inserted fragments was variable among the transformants, resulting in high yet uneven levels of transgene expression. In striking contrast, when the authentic URA5.3 gene (URACm-Cm) was used as a selection marker, efficient single-copy insertion was observed at the targeted locus. Thus, we have successfully established a highly reliable and reproducible method for gene targeting in C. Merolae. Our method will be applicable to a number of genetic manipulations in this organism, including targeted gene disruption, replacement and tagging.
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Mitochondrial Localization of Ferrochelatase in a Red Alga Cyanidioschyzon Merolae
Plant & cell physiology, 2013Co-Authors: Satoru Watanabe, Mio Ohnuma, Mitsumasa Hanaoka, Yusaku Ohba, Tomohiro Ono, Hirofumi Yoshikawa, Shigeru Taketani, Kan TanakaAbstract:Ferrochelatase (FECH) is an essential enzyme for the final step of heme biosynthesis. In green plants, its activity has been reported in both plastids and mitochondria. However, the precise subcellular localization of FECH remains uncertain. In this study, we analyzed the localization of FECH in the unicellular red alga, Cyanidioschyzon Merolae. Immunoblot and enzyme activity analyses of subcellular fractions localized little FECH in the plastid. In addition, immunofluorescence microscopy identified that both intrinsic and hemagglutinin (HA)-tagged FECH are localized in the mitochondrion. We therefore conclude that FECH is localized in the mitochondrion in C. Merolae.
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phototaxis in the unicellular red algae Cyanidioschyzon Merolae and cyanidium caldarium
Cytologia, 2011Co-Authors: Mio Ohnuma, Osami Misumi, Tsuneyoshi KuroiwaAbstract:Phototaxis of 2 cyanidiaceae, Cyanidioschyzon Merolae and Cyanidium caldarium, was studied by population experiments. We found that cells of both C. Merolae and C. caldarium moved towards light in liquid medium, but that the degree of migration was quite different. When laterally illuminated, most of the C. Merolae cells moved towards light at a velocity of 0.27 mm/h. In contrast, only a small proportion of C. caldarium cells showed migration towards light and most of the cells remained dispersed. The exterior cell surface of C. Merolae was observed by scanning electron microscopy. It appeared thick and flexible enough to enable crawling movement.
Takayuki Fujiwara - One of the best experts on this subject based on the ideXlab platform.
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escrt machinery mediates cytokinetic abscission in the unicellular red alga Cyanidioschyzon Merolae
Frontiers in Cell and Developmental Biology, 2020Co-Authors: Fumi Yagisawa, Shin-ya Miyagishima, Takayuki Fujiwara, Nobuko Sumiya, Yuki Kobayashi, Tokiaki Takemura, Soichi Nakamura, Yuuta ImotoAbstract:In many eukaryotes, cytokinesis proceeds in two successive steps: first, ingression of the cleavage furrow and second, abscission of the intercellular bridge. In animal cells, the actomyosin contractile ring is involved in the first step, while the endosomal sorting complex required for transport (ESCRT), which participates in various membrane fusion/fission events, mediates the second step. Intriguingly, in archaea, ESCRT is involved in cytokinesis, raising the hypothesis that the function of ESCRT in eukaryotic cytokinesis descended from the archaeal ancestor. In eukaryotes other than animals, the roles of ESCRT in cytokinesis have been poorly understood. To explore the primordial core mechanisms for eukaryotic cytokinesis, we investigated ESCRT functions in the unicellular red alga Cyanidioschyzon Merolae that diverged early in eukaryotic evolution. C. Merolae provides an excellent experimental system. The cell has a simple organelle composition. The genome (16.5 Mb, 5335 genes) has been completely sequenced, transformation methods are established, and the cell cycle is synchronized by a light and dark cycle. Similar to animal and fungal cells, C. Merolae cells divide through furrowing at the division site followed by abscission of the intercellular bridge. However, they lack an actomyosin contractile ring. The proteins that comprise ESCRT-I–IV, the four subcomplexes of ESCRT, are partially conserved in C. Merolae. Immunofluorescence of native or tagged proteins localized the homologs of the five ESCRT-III components [charged multivesicular body protein (CHMP) 1, 2, and 4–6], apoptosis-linked gene-2-interacting protein X (ALIX), the ESCRT-III adapter, and the main ESCRT-IV player vacuolar protein sorting (VPS) 4, to the intercellular bridge. In addition, ALIX was enriched around the cleavage furrow early in cytokinesis. When the ESCRT function was perturbed by expressing dominant-negative VPS4, cells with an elongated intercellular bridge accumulated, a phenotype resulting from abscission failure. Our results showed that ESCRT mediates cytokinetic abscission in C. Merolae. The fact that ESCRT plays a role in cytokinesis in archaea, animals, and early diverged alga C. Merolae supports the hypothesis that the function of ESCRT in cytokinesis descended from archaea to a common ancestor of eukaryotes.
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Day/Night Separation of Oxygenic Energy Metabolism and Nuclear DNA Replication in the Unicellular Red Alga Cyanidioschyzon Merolae
'American Society for Microbiology', 2019Co-Authors: Shin-ya Miyagishima, Nobuko Sumiya, Atsuko Era, Tomohisa Hasunuma, Mami Matsuda, Shunsuke Hirooka, Akihiko Kondo, Takayuki FujiwaraAbstract:ABSTRACT The transition from G1 to S phase and subsequent nuclear DNA replication in the cells of many species of eukaryotic algae occur predominantly during the evening and night in the absence of photosynthesis; however, little is known about how day/night changes in energy metabolism and cell cycle progression are coordinated and about the advantage conferred by the restriction of S phase to the night. Using a synchronous culture of the unicellular red alga Cyanidioschyzon Merolae, we found that the levels of photosynthetic and respiratory activities peak during the morning and then decrease toward the evening and night, whereas the pathways for anaerobic consumption of pyruvate, produced by glycolysis, are upregulated during the evening and night as reported recently in the green alga Chlamydomonas reinhardtii. Inhibition of photosynthesis by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) largely reduced respiratory activity and the amplitude of the day/night rhythm of respiration, suggesting that the respiratory rhythm depends largely on photosynthetic activity. Even when the timing of G1/S-phase transition was uncoupled from the day/night rhythm by depletion of retinoblastoma-related (RBR) protein, the same patterns of photosynthesis and respiration were observed, suggesting that cell cycle progression and energy metabolism are regulated independently. Progression of the S phase under conditions of photosynthesis elevated the frequency of nuclear DNA double-strand breaks (DSB). These results suggest that the temporal separation of oxygenic energy metabolism, which causes oxidative stress, from nuclear DNA replication reduces the risk of DSB during cell proliferation in C. Merolae. IMPORTANCE Eukaryotes acquired chloroplasts through an endosymbiotic event in which a cyanobacterium or a unicellular eukaryotic alga was integrated into a previously nonphotosynthetic eukaryotic cell. Photosynthesis by chloroplasts enabled algae to expand their habitats and led to further evolution of land plants. However, photosynthesis causes greater oxidative stress than mitochondrion-based respiration. In seed plants, cell division is restricted to nonphotosynthetic meristematic tissues and populations of photosynthetic cells expand without cell division. Thus, seemingly, photosynthesis is spatially sequestrated from cell proliferation. In contrast, eukaryotic algae possess photosynthetic chloroplasts throughout their life cycle. Here we show that oxygenic energy conversion (daytime) and nuclear DNA replication (night time) are temporally sequestrated in C. Merolae. This sequestration enables “safe” proliferation of cells and allows coexistence of chloroplasts and the eukaryotic host cell, as shown in yeast, where mitochondrial respiration and nuclear DNA replication are temporally sequestrated to reduce the mutation rate
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Day/Night Separation of Oxygenic Energy Metabolism and Nuclear DNA Replication in the Unicellular Red Alga Cyanidioschyzon Merolae
'American Society for Microbiology', 2019Co-Authors: Shin-ya Miyagishima, Nobuko Sumiya, Atsuko Era, Tomohisa Hasunuma, Mami Matsuda, Shunsuke Hirooka, Akihiko Kondo, Takayuki FujiwaraAbstract:Eukaryotes acquired chloroplasts through an endosymbiotic event in which a cyanobacterium or a unicellular eukaryotic alga was integrated into a previously nonphotosynthetic eukaryotic cell. Photosynthesis by chloroplasts enabled algae to expand their habitats and led to further evolution of land plants. However, photosynthesis causes greater oxidative stress than mitochondrion-based respiration. In seed plants, cell division is restricted to nonphotosynthetic meristematic tissues and populations of photosynthetic cells expand without cell division. Thus, seemingly, photosynthesis is spatially sequestrated from cell proliferation. In contrast, eukaryotic algae possess photosynthetic chloroplasts throughout their life cycle. Here we show that oxygenic energy conversion (daytime) and nuclear DNA replication (night time) are temporally sequestrated in C. Merolae. This sequestration enables “safe” proliferation of cells and allows coexistence of chloroplasts and the eukaryotic host cell, as shown in yeast, where mitochondrial respiration and nuclear DNA replication are temporally sequestrated to reduce the mutation rate.The transition from G1 to S phase and subsequent nuclear DNA replication in the cells of many species of eukaryotic algae occur predominantly during the evening and night in the absence of photosynthesis; however, little is known about how day/night changes in energy metabolism and cell cycle progression are coordinated and about the advantage conferred by the restriction of S phase to the night. Using a synchronous culture of the unicellular red alga Cyanidioschyzon Merolae, we found that the levels of photosynthetic and respiratory activities peak during the morning and then decrease toward the evening and night, whereas the pathways for anaerobic consumption of pyruvate, produced by glycolysis, are upregulated during the evening and night as reported recently in the green alga Chlamydomonas reinhardtii. Inhibition of photosynthesis by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) largely reduced respiratory activity and the amplitude of the day/night rhythm of respiration, suggesting that the respiratory rhythm depends largely on photosynthetic activity. Even when the timing of G1/S-phase transition was uncoupled from the day/night rhythm by depletion of retinoblastoma-related (RBR) protein, the same patterns of photosynthesis and respiration were observed, suggesting that cell cycle progression and energy metabolism are regulated independently. Progression of the S phase under conditions of photosynthesis elevated the frequency of nuclear DNA double-strand breaks (DSB). These results suggest that the temporal separation of oxygenic energy metabolism, which causes oxidative stress, from nuclear DNA replication reduces the risk of DSB during cell proliferation in C. Merolae
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Development of a Heat-Shock Inducible Gene Expression System in the Red Alga Cyanidioschyzon Merolae
2016Co-Authors: Nobuko Sumiya, Takayuki Fujiwara, Yusuke Kobayashi, Osami MisumiAbstract:The cell of the unicellular red alga Cyanidioschyzon Merolae contains a single chloroplast and mitochondrion, the division of which is tightly synchronized by a light/dark cycle. The genome content is extremely simple, with a low level of genetic redundancy, in photosynthetic eukaryotes. In addition, transient transformation and stable transformation by homologous recombination have been reported. However, for molecular genetic analyses of phenomena that are essential for cellular growth and survival, inducible gene expression/suppression systems are needed. Here, we report the development of a heat-shock inducible gene expression system in C. Merolae. CMJ101C, encoding a small heat shock protein, is transcribed only when cells are exposed to an elevated temperature. Using a superfolder GFP as a reporter protein, the 200-bp upstream region of CMJ101C orf was determined to be the optimal promoter for heat-shock induction. The optimal temperature to induce expression is 50uC, at which C. Merolae cells are able to proliferate. At least a 30-min heat shock is required for the expression of a protein of interest and a 60-min heat shock yields the maximum level of protein expression. After the heat shock, the mRNA level decreases rapidly. As an example of the system, the expression of a dominant negative form of chloroplast division DRP5B protein, which has a mutation in the GTPase domain, was induced. Expression of the dominant negative DRP5B resulted in the appearance of aberrant-shaped cells in which two daughter chloroplasts and the cells are still connected by a small DRP5B positive tube-like structure. This result suggests that the dominant negativ
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algae sense exact temperatures small heat shock proteins are expressed at the survival threshold temperature in Cyanidioschyzon Merolae and chlamydomonas reinhardtii
Genome Biology and Evolution, 2014Co-Authors: Yusuke Kobayashi, Tsuneyoshi Kuroiwa, Takayuki Fujiwara, Naomi Harada, Yoshiki Nishimura, Takafumi Saito, Mami Nakamura, Osami MisumiAbstract:The primitive red alga Cyanidioschyzon Merolae inhabits acidic hot springs and shows robust resistance to heat shock treatments up to 63 °C. Microarray analysis was performed to identify the key genes underlying the high temperature tolerance of this organism. Among the upregulated genes that were identified, we focused on two small heat shock proteins (sHSPs) that belong to a unique class of HSP families. These two genes are located side by side in an inverted repeat orientation on the same chromosome and share a promoter. These two genes were simultaneously and rapidly upregulated in response to heat shock treatment (>1,000-fold more than the control). Interestingly, upregulation appeared to be triggered not by a difference in temperatures, but rather by the absolute temperature. Similar sHSP structural genes have been reported in the green alga Chlamydomonas reinhardtii, but the threshold temperature for the expression of these sHSP-encoding genes in Ch. reinhardtii was different from the threshold temperature for the expression of the sHSP genes from Cy. Merolae. These results indicate the possible importance of an absolute temperature sensing system in the evolution and tolerance of high-temperature conditions among unicellular microalgae.
Fumi Yagisawa - One of the best experts on this subject based on the ideXlab platform.
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escrt machinery mediates cytokinetic abscission in the unicellular red alga Cyanidioschyzon Merolae
Frontiers in Cell and Developmental Biology, 2020Co-Authors: Fumi Yagisawa, Shin-ya Miyagishima, Takayuki Fujiwara, Nobuko Sumiya, Yuki Kobayashi, Tokiaki Takemura, Soichi Nakamura, Yuuta ImotoAbstract:In many eukaryotes, cytokinesis proceeds in two successive steps: first, ingression of the cleavage furrow and second, abscission of the intercellular bridge. In animal cells, the actomyosin contractile ring is involved in the first step, while the endosomal sorting complex required for transport (ESCRT), which participates in various membrane fusion/fission events, mediates the second step. Intriguingly, in archaea, ESCRT is involved in cytokinesis, raising the hypothesis that the function of ESCRT in eukaryotic cytokinesis descended from the archaeal ancestor. In eukaryotes other than animals, the roles of ESCRT in cytokinesis have been poorly understood. To explore the primordial core mechanisms for eukaryotic cytokinesis, we investigated ESCRT functions in the unicellular red alga Cyanidioschyzon Merolae that diverged early in eukaryotic evolution. C. Merolae provides an excellent experimental system. The cell has a simple organelle composition. The genome (16.5 Mb, 5335 genes) has been completely sequenced, transformation methods are established, and the cell cycle is synchronized by a light and dark cycle. Similar to animal and fungal cells, C. Merolae cells divide through furrowing at the division site followed by abscission of the intercellular bridge. However, they lack an actomyosin contractile ring. The proteins that comprise ESCRT-I–IV, the four subcomplexes of ESCRT, are partially conserved in C. Merolae. Immunofluorescence of native or tagged proteins localized the homologs of the five ESCRT-III components [charged multivesicular body protein (CHMP) 1, 2, and 4–6], apoptosis-linked gene-2-interacting protein X (ALIX), the ESCRT-III adapter, and the main ESCRT-IV player vacuolar protein sorting (VPS) 4, to the intercellular bridge. In addition, ALIX was enriched around the cleavage furrow early in cytokinesis. When the ESCRT function was perturbed by expressing dominant-negative VPS4, cells with an elongated intercellular bridge accumulated, a phenotype resulting from abscission failure. Our results showed that ESCRT mediates cytokinetic abscission in C. Merolae. The fact that ESCRT plays a role in cytokinesis in archaea, animals, and early diverged alga C. Merolae supports the hypothesis that the function of ESCRT in cytokinesis descended from archaea to a common ancestor of eukaryotes.
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Evolutionary significance of the ring-like plastid nucleus in the primitive red alga Cyanidioschyzon Merolae as revealed by drying
Protoplasma, 2020Co-Authors: Tsuneyoshi Kuroiwa, Mio Ohnuma, Yuuta Imoto, Fumi Yagisawa, Osami Misumi, Noriko Nagata, Haruko KuroiwaAbstract:Primary plastids originated from a free-living cyanobacterial ancestor and possess their own genomes—probably a few DNA copies. These genomes, which are organized in centrally located plastid nuclei (CN-type pt-nuclei), are produced from preexisting plastids by binary division. Ancestral algae with a CN-type pt-nucleus diverged and evolved into two basal eukaryotic lineages: red algae with circular (CL-type) pt-nuclei and green algae with scattered small (SN-type) pt-nuclei. Although the molecular dynamics of pt-nuclei in green algae and plants are now being analyzed, the process of the conversion of the original algae with a CN-type pt-nucleus to red algae with a CL-type one has not been studied. Here, we show that the CN-type pt-nucleus in the primitive red alga Cyanidioschyzon Merolae can be changed to the CL-type by application of drying to produce slight cell swelling. This result implies that CN-type pt-nuclei are produced by compact packing of CL-type ones, which suggests that a C. Merolae– like alga was the original progenitor of the red algal lineage. We also observed that the CL-type pt-nucleus has a chain-linked bead-like structure. Each bead is most likely a small unit of DNA, similar to CL-type pt-nuclei in brown algae. Our results thus suggest a C. Merolae –like alga as the candidate for the secondary endosymbiont of brown algae.
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mitotic inheritance of endoplasmic reticulum in the primitive red alga Cyanidioschyzon Merolae
Protoplasma, 2012Co-Authors: Yuuta Imoto, Fumi Yagisawa, Haruko Kuroiwa, Takayuki Fujiwara, Keiji Nishida, Tsuneyoshi KuroiwaAbstract:Endoplasmic reticulum (ER) is a major site for secretory protein folding and lipid synthesis. Since ER cannot be synthesized de novo, it must be inherited during the cell cycle. Studying ER inheritance can however be difficult because the ER of typical plant and animal cells is morphologically complex. Therefore, our study used Cyanidioschyzon Merolae, a species that has a simple ER structure, to investigate the inheritance of this organelle. Using immunofluorescence microscopy, we demonstrated that C. Merolae contains a nuclear ER (nuclear envelope) and a small amount of peripheral ER extending from the nuclear ER. During mitosis, the nuclear ER became dumbbell-shaped and underwent division. Peripheral ER formed ring-like structures during the G1 and S phases, and extended toward the mitochondria and cell division planes during the M phase. These observations indicated that C. Merolae undergoes closed mitosis, whereby the nuclear ER does not diffuse, and the peripheral ER contains cell cycle-specific structures.
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chloroplasts divide by contraction of a bundle of nanofilaments consisting of polyglucan
Science, 2010Co-Authors: Yamato Yoshida, Mio Ohnuma, Yuuta Imoto, Fumi Yagisawa, Osami Misumi, Haruko Kuroiwa, Takayuki Fujiwara, Masaki Yoshida, Shunsuke Hirooka, Kazunobu MatsushitaAbstract:In chloroplast division, the plastid-dividing (PD) ring is a main structure of the PD machinery and is a universal structure in the plant kingdom. However, the components and formation of the PD ring have been enigmatic. By proteomic analysis of PD machineries isolated from Cyanidioschyzon Merolae, we identified the glycosyltransferase protein plastid-dividing ring 1 (PDR1), which constructs the PD ring and is widely conserved from red alga to land plants. Electron microscopy showed that the PDR1 protein forms a ring with carbohydrates at the chloroplast-division site. Fluorometric saccharide ingredient analysis of purified PD ring filaments showed that only glucose was included, and down-regulation of PDR1 impaired chloroplast division. Thus, the chloroplasts are divided by the PD ring, which is a bundle of PDR1-mediated polyglucan filaments.
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periodic gene expression patterns during the highly synchronized cell nucleus and organelle division cycles in the unicellular red alga Cyanidioschyzon Merolae
DNA Research, 2009Co-Authors: Takayuki Fujiwara, Fumi Yagisawa, Osami Misumi, Sousuke Imamura, Kousuke Tashiro, Yamato Yoshida, Keiji Nishida, Masaki Yoshida, Toshiyuki Mori, Kan TanakaAbstract:Previous cell cycle studies have been based on cell-nuclear proliferation only. Eukaryotic cells, however, have double membranes-bound organelles, such as the cell nucleus, mitochondrion, plastids and single-membrane-bound organelles such as ER, the Golgi body, vacuoles (lysosomes) and microbodies. Organelle proliferations, which are very important for cell functions, are poorly understood. To clarify this, we performed a microarray analysis during the cell cycle of Cyanidioschyzon Merolae. C. Merolae cells contain a minimum set of organelles that divide synchronously. The nuclear, mitochondrial and plastid genomes were completely sequenced. The results showed that, of 158 genes induced during the S or G2-M phase, 93 were known and contained genes related to mitochondrial division, ftsZ1-1, ftsz1-2 and mda1, and plastid division, ftsZ2-1, ftsZ2-2 and cmdnm2. Moreover, three genes, involved in vesicle trafficking between the single-membrane organelles such as vps29 and the Rab family protein, were identified and might be related to partitioning of single-membrane-bound organelles. In other genes, 46 were hypothetical and 19 were hypothetical conserved. The possibility of finding novel organelle division genes from hypothetical and hypothetical conserved genes in the S and G2-M expression groups is discussed.
