Published Papers - Yagisawa Fumi

A fusion protein of polyphosphate kinase 1 (PPK1) and a Nudix hydrolase is involved in inorganic polyphosphate accumulation in the unicellular red alga Cyanidioschyzon merolae.

Yagisawa F, Fujiwara T, Yamashita S, Hirooka S, Tamashiro K, Izumi J, Kanesaki Y, Onuma R, Misumi O, Nakamura S, Yoshikawa H, Kuroiwa H, Kuroiwa T, Miyagishima SY

Plant molecular biology ( Springer Science and Business Media LLC )  115 ( 1 ) 9   2024.12 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Development of a rapamycin-inducible protein-knockdown system in the unicellular red alga Cyanidioschyzon merolae.

Fujiwara T, Hirooka S, Yamashita S, Yagisawa F, Miyagishima SY

Plant physiology ( Oxford University Press (OUP) )  196 ( 1 ) 77 - 94   2024.06 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Abstract An inducible protein-knockdown system is highly effective for investigating the functions of proteins and mechanisms essential for the survival and growth of organisms. However, this technique is not available in photosynthetic eukaryotes. The unicellular red alga Cyanidioschyzon merolae possesses a very simple cellular and genomic architecture and is genetically tractable but lacks RNA interference machinery. In this study, we developed a protein-knockdown system in this alga. The constitutive system utilizes the destabilizing activity of the FK506-binding protein 12 (FKBP12)-rapamycin-binding (FRB) domain of human target of rapamycin kinase or its derivatives to knock down target proteins. In the inducible system, rapamycin treatment induces the heterodimerization of the human FRB domain fused to the target proteins with the human FKBP fused to S-phase kinase-associated protein 1 or Cullin 1, subunits of the SCF E3 ubiquitin ligase. This results in the rapid degradation of the target proteins through the ubiquitin-proteasome pathway. With this system, we successfully degraded endogenous essential proteins such as the chloroplast division protein dynamin-related protein 5B and E2 transcription factor, a regulator of the G1/S transition, within 2 to 3 h after rapamycin administration, enabling the assessment of resulting phenotypes. This rapamycin-inducible protein-knockdown system contributes to the functional analysis of genes whose disruption leads to lethality.

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Complete mitochondrial and chloroplast DNA sequences of the freshwater green microalga Medakamo hakoo.

Takusagawa M, Misumi O, Nozaki H, Kato S, Maruyama S, Tsujimoto-Inui Y, Yagisawa F, Ohnuma M, Kuroiwa H, Kuroiwa T, Matsunaga S

Genes & genetic systems ( 日本遺伝学会 )  98 ( 6 ) 353 - 360   2024.02

Type of publication: Research paper (scientific journal)

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<p>We report the complete organellar genome sequences of an ultrasmall green alga, <i>Medakamo hakoo</i> strain M-hakoo 311, which has the smallest known nuclear genome in freshwater green algae. <i>Medakamo hakoo</i> has 90.8-kb chloroplast and 36.5-kb mitochondrial genomes containing 80 and 33 putative protein-coding genes, respectively. The mitochondrial genome is the smallest in the Trebouxiophyceae algae studied so far. The GC content of the nuclear genome is 73%, but those of chloroplast and mitochondrial genomes are 41% and 35%, respectively. Codon usages in the organellar genomes have a different tendency from that in the nuclear genome. The organellar genomes have unique characteristics, such as the biased encoding of mitochondrial genes on a single strand and the absence of operon structures in chloroplast ribosomal genes. <i>Medakamo hakoo</i> will be helpful for understanding the evolution of the organellar genome and the regulation of gene expression in chloroplasts and mitochondria.</p>

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Genomic analysis of an ultrasmall freshwater green alga, Medakamo hakoo.

Kato S, Misumi O, Maruyama S, Nozaki H, Tsujimoto-Inui Y, Takusagawa M, Suzuki S, Kuwata K, Noda S, Ito N, Okabe Y, Sakamoto T, Yagisawa F, Matsunaga TM, Matsubayashi Y, Yamaguchi H, Kawachi M, Kuroiwa H, Kuroiwa T, Matsunaga S

Communications biology ( Springer Science and Business Media LLC )  6 ( 1 ) 89   2023.01 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Abstract Ultrasmall algae have attracted the attention of biologists investigating the basic mechanisms underlying living systems. Their potential as effective organisms for producing useful substances is also of interest in bioindustry. Although genomic information is indispensable for elucidating metabolism and promoting molecular breeding, many ultrasmall algae remain genetically uncharacterized. Here, we present the nuclear genome sequence of an ultrasmall green alga of freshwater habitats, Medakamo hakoo. Evolutionary analyses suggest that this species belongs to a new genus within the class Trebouxiophyceae. Sequencing analyses revealed that its genome, comprising 15.8 Mbp and 7629 genes, is among the smallest known genomes in the Viridiplantae. Its genome has relatively few genes associated with genetic information processing, basal transcription factors, and RNA transport. Comparative analyses revealed that 1263 orthogroups were shared among 15 ultrasmall algae from distinct phylogenetic lineages. The shared gene sets will enable identification of genes essential for algal metabolism and cellular functions.

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Complete Mitochondrial and Plastid DNA Sequences of the Freshwater Green Microalga Medakamo hakoo

Mari Takusagawa, Shoichi Kato, Sachihiro Matsunaga, Shinichiro Maruyama, Yayoi Tsujimoto-Inui, Hisayoshi Nozaki, Fumi Yagisawa, Mio Ohnuma, Haruko Kuroiwa, Tsuneyoshi Kuroiwa, Osami Misumi

bioRxiv ( Cold Spring Harbor Laboratory )    2021.07

Type of publication: Research paper (scientific journal)

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<jats:p>Here we report the complete organellar genome sequences of <jats:italic>Medakamo hakoo</jats:italic>, a green alga identified in freshwater in Japan. It has 90.8-kb plastid and 36.5-kb mitochondrial genomes containing 80 and 33 putative protein coding genes, respectively, representing the smallest organellar genome among currently known core Trebouxiophyceae.</jats:p>

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Smooth Loop-Like Mitochondrial Nucleus in the Primitive Red Alga <i>Cyanidioschyzon merolae</i> Revealed by Drying Treatment

Kuroiwa Tsuneyoshi, Yagisawa Fumi, Fujiwara Takayuki, Misumi Osami, Nagata Noriko, Imoto Yuuta, Yoshida Yamato, Mogi Yuko, Miyagishima Shin-ya, Kuroiwa Haruko

ＣＹＴＯＬＯＧＩＡ ( 公益財団法人 日本メンデル協会 )  86 ( 1 ) 89 - 96   2021.03 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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<p>It is thought that mitochondria were generated by the symbiosis of autonomous α-proteobacteria and a eukaryote-like organism derived from an archaeon of the species <i>Sulfolobus</i>. Soon after the symbiosis, most of the genome of the α-proteobacterium, which was required for autonomy, was lost. Many genes were transferred into the host genome. However, a small amount of DNA—the mitochondrial genome (mt-genome, mtDNA)—remained in the symbiotic organelle. The primitive eukaryotic cells increased the mtDNA copy number and formed a mitochondrial nucleus (mt-nucleus). The primitive unicellular eukaryote evolved into organisms with one mitochondrion containing multiple mtDNA copies per cell, and organisms with multiple mitochondria with a small number of mtDNA copies in each cell. There have been many studies on the mitochondria and mt-genomes of amoeba, plants, and animals which contain many mitochondria per cell, but only a few studies have reported morphological characteristics of the mitochondria and their genomes in primitive unicellular organisms that have only a single mitochondrion per cell. Here, we show that centrally located mt-nuclei in the primitive red alga <i>Cyanidioschyzon merolae</i> form smooth rings following the application of a drying method that produces slight cell swelling. We discuss regulatory mechanisms for genome function in endosymbiotic organelles on the basis of the differences between the copy number of mtDNA in smooth-ring shaped mt-nuclei and plastid DNA in bead-shaped plastid nuclei.</p>

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Evolutionary significance of the ring-like plastid nucleus in the primitive red alga Cyanidioschyzon merolae as revealed by drying.

Kuroiwa T, Ohnuma M, Imoto Y, Yagisawa F, Misumi O, Nagata N, Kuroiwa H

Protoplasma ( Protoplasma )  257 ( 4 ) 1069 - 1078   2020.07 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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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 Karyotype of the Primitive Red Alga <i>Cyanidioschyzon merolae</i> 10D

Kuroiwa Tsuneyoshi, Kuroiwa Haruko, Yagisawa Fumi, Fujiwara Takayuki, Inui Yayoi, M. Matsunaga Tomoko, Katoi Shoichi, Matsunaga Sachihiro, Nagata Noriko, Imoto Yuuta

ＣＹＴＯＬＯＧＩＡ ( 公益財団法人 日本メンデル協会 )  85 ( 2 ) 107 - 113   2020 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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<p>It is important to understand how a single circular chromosome in the prokaryotic nucleus evolved into multiple linear chromosomes in the eukaryotic nucleus. In most eukaryotic cells that have ＞∼15 Mbp of genomic DNA, chromosomes remain condensed through all the mitotic phases. Therefore, we observed nuclei of primitive organisms in which linear chromosomes had not been observed previously using conventional methods. Cells of the primitive red alga <i>Cyanidioschyzon merolae</i>, having a genome size of 16.5 Mbp, have been used to study the division of organelles, such as mitochondria, chloroplasts, and peroxisomes. However, morphologically condensed chromosomes have never been observed during mitotic metaphase. Recently, we demonstrated that plastid nuclei are swollen and change from a spherical to a ring shape after being subjected to the glutaraldehyde-fixed-drying method. Using a modified method, we visualized mitotic chromosomes in <i>C. merolae</i> cells. Chromosomal condensation occurred just after the chloroplast division when cells enter metaphase. Thus, chromosomal separation in <i>C. merolae</i> cells likely occurs in a manner similar to that of typical eukaryotic cells. However, mitotic condensed chromosomes were not observed in the primitive green alga <i>Medakamo hakoo</i>, having a genome size of 8 Mbp. Thus, the results support the use of <i>C. merolae</i> as a model for eukaryotic cell analyses.</p>

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ESCRT Machinery Mediates Cytokinetic Abscission in the Unicellular Red Alga <i>Cyanidioschyzon merolae</i>.

Yagisawa F, Fujiwara T, Takemura T, Kobayashi Y, Sumiya N, Miyagishima SY, Nakamura S, Imoto Y, Misumi O, Tanaka K, Kuroiwa H, Kuroiwa T

Frontiers in cell and developmental biology ( Frontiers in Cell and Developmental Biology )  8   169 - 169   2020 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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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 in animals, the roles of ESCRT in cytokinesis are 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 by 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 show 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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Sphingolipids activate the endoplasmic reticulum stress surveillance pathway.

Piña F, Yagisawa F, Obara K, Gregerson JD, Kihara A, Niwa M

The Journal of cell biology   217 ( 2 ) 495 - 505   2018.01 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Proper inheritance of functional organelles is vital to cell survival. In the budding yeast, Saccharomyces cerevisiae, the endoplasmic reticulum (ER) stress surveillance (ERSU) pathway ensures that daughter cells inherit a functional ER. Here, we show that the ERSU pathway is activated by phytosphingosine (PHS), an early biosynthetic sphingolipid. Multiple lines of evidence support this: (1) Reducing PHS levels with myriocin diminishes the ability of cells to induce ERSU phenotypes. (2) Aureobasidin A treatment, which blocks conversion of early intermediates to downstream complex sphingolipids, induces ERSU. (3) orm1Δorm2Δ cells, which up-regulate PHS, show an ERSU response even in the absence of ER stress. (4) Lipid analyses confirm that PHS levels are indeed elevated in ER-stressed cells. (5) Lastly, the addition of exogenous PHS is sufficient to induce all ERSU phenotypes. We propose that ER stress elevates PHS, which in turn activates the ERSU pathway to ensure future daughter-cell viability.

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Glycosyltransferase MDR1 assembles a dividing ring for mitochondrial proliferation comprising polyglucan nanofilaments.

Yoshida Y, Kuroiwa H, Shimada T, Yoshida M, Ohnuma M, Fujiwara T, Imoto Y, Yagisawa F, Nishida K, Hirooka S, Misumi O, Mogi Y, Akakabe Y, Matsushita K, Kuroiwa T

Proceedings of the National Academy of Sciences of the United States of America ( NATL ACAD SCIENCES )  114 ( 50 ) 13284 - 13289   2017.12 [ Peer Review Accepted ]

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Mitochondria, which evolved from a free-living bacterial ancestor, contain their own genomes and genetic systems and are produced from preexisting mitochondria by binary division. The mitochondrion-dividing (MD) ring is the main skeletal structure of the mitochondrial division machinery. However, the assembly mechanism and molecular identity of the MD ring are unknown. Multi-omics analysis of isolated mitochondrial division machinery from the unicellular alga Cyanidioschyzon merolae revealed an uncharacterized glycosyltransferase, MITOCHONDRION-DIVIDING RING1 (MDR1), which is specifically expressed during mitochondrial division and forms a single ring at the mitochondrial division site. Nanoscale imaging using immunoelectron microscopy and componential analysis demonstrated that MDR1 is involved in MD ring formation and that the MD ring filaments are composed of glycosylated MDR1 and polymeric glucose nanofilaments. Down-regulation of MDR1 strongly interrupted mitochondrial division and obstructed MD ring assembly. Taken together, our results suggest that MDR1 mediates the synthesis of polyglucan nanofilaments that assemble to form the MD ring. Given that a homolog of MDR1 performs similar functions in chloroplast division, the establishment of MDR1 family proteins appears to have been a singular, crucial event for the emergence of endosymbiotic organelles.

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Intracellular Structure of the Unicellular Red Alga <i>Cyanidioschyzon merolae</i> in Response to Phosphate Depletion and Resupplementation

Yagisawa Fumi, Kuroiwa Haruko, Fujiwara Takayuki, Kuroiwa Tsuneyoshi

ＣＹＴＯＬＯＧＩＡ ( 財団法人 日本メンデル協会 )  81 ( 3 ) 341 - 347   2016 [ Peer Review Accepted ]

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<p>In algae, vacuolar polyphosphate stores phosphate, regulates osmotic pressure and cytosolic pH, and buffers metallic cations. However, its metabolism is not well understood. <i>Cyanidioschyzon merolae</i> cells contain four to five vacuoles storing polyphosphate whose degradation and synthesis are induced by phosphate deprivation and resupplementation, respectively. To understand polyphosphate metabolism, we observed <i>C. merolae</i> cells cultured in different phosphate conditions by electron microscopy. In the phosphate-containing medium, the vacuoles contained electron-dense substances presumably consisting of polyphosphate and metallic cations. When phosphate was depleted, the vacuoles lost the electron-dense substances, and lipid bodies in close contact with vacuoles and starch grains appeared in the cytoplasm. When phosphate was added again, small, electron-dense granules or vesicles appeared in the cytoplasm. Some of them were in contact with the vacuoles and had been taken into the vacuolar lumen, suggesting the possibility that polyphosphate is synthesized and transported to the vacuoles in these structures.</p>

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Genome Size of the Ultrasmall Unicellular Freshwater Green Alga, Medakamo hakoo 311, as Determined by Staining with 4′,6-Diamidino-2-phenylindole after Microwave Oven Treatments: II. Comparison with Cyanidioschyzon merolae, Saccharomyces cerevisiae (n, 2n), and Chlorella variabilis

Kuroiwa Tsuneyoshi, Ohnuma Mio, Imoto Yuuta, Misumi Osami, Nagata Noriko, Miyakawa Isamu, Fujishima Masahiro, Yagisawa Fumi, Kuroiwa Haruko

CYTOLOGIA ( 財団法人 日本メンデル協会 )  81 ( 1 ) 69 - 76   2016 [ Peer Review Accepted ]

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Eukaryotic cells contain three types of nuclei: the cell nucleus, the mitochondrial nuclei (nucleoids), and plastid nuclei (nucleoids). Here, we report that the chlorophyte strain Medakamo hakoo 311 has the smallest cell nucleus genome among the free-living eukaryotes analyzed to date. The size of the nuclear genome was analyzed by staining with 4′,6-diamidino-2-phenylindole (DAPI), followed by fluorimetry using a video-intensified microscope photon-counting system (VIMPCS) and fluorescence microscopy. The exact genome size of M. hakoo remains elusive because of the low permeability of the thick algal cell wall to DAPI. To improve the DAPI staining method for M. hakoo, we added a microwave oven heating step. We examined the genome sizes of M. hakoo, Cyanidioschyzon merolae, Saccharomyces cerevisiae (n, 2n), and Chlorella variabilis 1 N by DAPI staining, which included a microwave oven heating step, followed by fluorimetry with the VIMPCS. The new method of DAPI staining that included a microwave oven heating step improved the staining efficiency of C. variabilis and M. hakoo nuclei, and slightly improved the staining efficiency of S. cerevisiae and C. merolae nuclei. Using well-stained samples and the cell nucleus genome size of C. merolae (16.5 Mb) as the standard, the cell nucleus genome sizes of M. hakoo, S. cerevisiae (n), and C. variabilis were determined as 9.2, 20.2, and 35.9 Mb, respectively. The results are consistent with previous reports that M. hakoo cell has the smallest genome among the free-living eukaryotes evaluated to date.

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