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

We report the complete organellar genome sequences of an ultrasmall green alga, Medakamo hakoo strain M-hakoo 311, which has the smallest known nuclear genome in freshwater green algae. Medakamo hakoo 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. Medakamo hakoo will be helpful for understanding the evolution of the organellar genome and the regulation of gene expression in chloroplasts and mitochondria.

Photo-controllable azobenzene microdroplets on an open surface and their application as transporters.

The control of droplet motion is a significant challenge, as there has been no simple method for effective manipulation. Utilizing light for the control of droplets offers a promising solution due to its non-contact nature and high degree of controllability. In this study, we present our findings on the translational motion of pre-photomelted droplets composed of azobenzene derivatives on a glass surface when exposed to UV and visible light sources from different directions. These droplets exhibited directional and continuous motion upon light irradiation and this motion was size-dependent. Only droplets with diameters less than 10 µm moved with a maximum velocity of 300 µm min-1. In addition, the direction of the movement was controllable by the direction of the light. The motion is driven by a change in contact angle, where UV or visible light switched the contact angle to approximately 50° or 35°, respectively. In addition, these droplets were also found to be capable carriers for fluorescent quantum dots. As such, droplets composed of photoresponsive molecules offer unique opportunities for designing novel light-driven open-surface microfluidic systems.

Photo-Induced Crawling Motion of Azobenzene Crystals on Modified Gold Surfaces.

Photo-induced crawling motion of a crystal of 3,3'-dimethylazobenzene (DMAB) on gold surfaces having different surface properties and various patterns was studied. DMAB crystals crawl continuously when exposed to UV and visible lights simultaneously from different directions. On a gold surface functionalized by a thiol having a hydroxyl group at the terminal (16-hydroxy-1-hexadecanethiol (HOC16SH)), the crystals crawled with a relatively high velocity (ca. 4 µm min-1), and they changed the crystal shape while keeping a distinct crystal face. On a gold surface functionalized by a thiol having an alkyl chain terminal (1-hexadecanethiol (C16SH)), the crawling was observed with a slower velocity (ca. 1.5 µm min-1). However, the shape of the crystals became a droplet-like shape soon after the irradiation started, and the shape persisted during the motion. Light intensity dependence of the crawling velocity of the droplet-like crystal on this surface showed that UV light has stronger dependence for the motion than the visible light. On a substrate with a stripe pattern of alternating C16SH-modified gold and hexadecyltrimethylsilane (HDTMS)-modified glass, crystals crawled only on the surface of the C16SH-modified gold, which may be due to the wettability hysteresis at the surface. On a substrate with a stripe pattern of HOC16SH-modified gold and HDTMS-modified glass, crystals were attracted to the gold side. On a gold substrate with a periodic pattern of different height (ca. 50 nm) but having a uniform treatment with C16SH, crystals crawled up and down the steps without significant disturbance at the boundary of the step. Therefore, wettability of the surface has a greater impact on controlling the motion of the crystal than the surface structure. The present results not only unveil the crawling behavior on various surfaces but also offer a guide to controlling the motion toward applications for novel carriage vehicles to transport molecules/objects on a surface.

Effect of Surface Properties on the Photo-Induced Crawling Motion of Azobenzene Crystals on Glass Surfaces.

Photo-induced crawling motion of a crystal of 3,3'-dimethylazobenzene (DMAB) on a glass substrate having different surface properties was studied. When exposed to UV and visible lights simultaneously from different directions, crystals crawl continuously on a glass surface. On a hydrophilic surface, the crystals crawled faster than those on other surfaces but crystals showed spreading while they moved. On hydrophobic surfaces, on the other hand, the crystals showed little shape change and slower crawling motion. The contact angles of the liquid phase of DMAB on surface-modified glass substrates showed positive correlation with the water contact angles. The interaction of melted azobenzene with glass surfaces plays an important role for the crawling motion. We proposed models to explain the asymmetric condition that leads to the directional motion. Specifically by considering the penetration length of UV and visible light sources, it was successfully shown that the depth of light penetration is different at the position of a crystal. This creates a nonequilibrium condition where melting and crystallization are predominant in the same crystal.

Efficient open cultivation of cyanidialean red algae in acidified seawater.

Microalgae possess high potential for producing pigments, antioxidants, and lipophilic compounds for industrial applications. However, their open pond cultures are often contaminated by other undesirable organisms, including their predators. In addition, the cost of using freshwater is relatively high, which limits the location and scale of cultivation compared with using seawater. It was previously shown that Cyanidium caldarium and Galdieria sulphuraria, but not Cyanidioschyzon merolae grew in media containing NaCl at a concentration equivalent to seawater. We found that the preculture of C. merolae in the presence of a moderate NaCl concentration enabled the cells to grow in the seawater-based medium. The cultivation of cyanidialean red algae in the seawater-based medium did not require additional pH buffering chemicals. In addition, the combination of seawater and acidic conditions reduced the risk of contamination by other organisms in the nonsterile open culture of C. merolae more efficiently than the acidic condition alone.

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

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.

Cyanidioschyzon merolae aurora kinase phosphorylates evolutionarily conserved sites on its target to regulate mitochondrial division.

The mitochondrion is an organelle that was derived from an endosymbiosis. Although regulation of mitochondrial growth by the host cell is necessary for the maintenance of mitochondria, it is unclear how this regulatory mechanism was acquired. To address this, we studied the primitive unicellular red alga Cyanidioschyzon merolae, which has the simplest eukaryotic genome and a single mitochondrion. Here we show that the C. merolae Aurora kinase ortholog CmAUR regulates mitochondrial division through phosphorylation of mitochondrial division ring components. One of the components, the Drp1 ortholog CmDnm1, has at least four sites phosphorylated by CmAUR. Depletion of the phosphorylation site conserved among eukaryotes induced defects such as mitochondrial distribution on one side of the cell. Taken together with the observation that human Aurora kinase phosphorylates Drp1 in vitro, we suggest that the phosphoregulation is conserved from the simplest eukaryotes to mammals, and was acquired at the primitive stage of endosymbiosis.

Negative phototactic behaviour of crystals on a glass surface.

Phototaxis of azobenzene crystals is demonstrated for the first time by using a simple setup. The crystals on a glass move away from the light source like living organisms during visible light irradiation. The motion is driven by repeated melting and recrystallization associated with trans↔cis isomerization.

Dynamics of the nucleoside diphosphate kinase protein DYNAMO2 correlates with the changes in the global GTP level during the cell cycle of Cyanidioschyzon merolae.

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.

Onsite GTP fuelling via DYNAMO1 drives division of mitochondria and peroxisomes.

Mitochondria and peroxisomes proliferate by division. During division, a part of their membrane is pinched off by constriction of the ring-shaped mitochondrial division (MD) and peroxisome-dividing (POD) machinery. This constriction is mediated by a dynamin-like GTPase Dnm1 that requires a large amount of GTP as an energy source. Here, via proteomics of the isolated division machinery, we show that the 17-kDa nucleoside diphosphate kinase-like protein, dynamin-based ring motive-force organizer 1 (DYNAMO1), locally generates GTP in MD and POD machineries. DYNAMO1 is widely conserved among eukaryotes and colocalizes with Dnm1 on the division machineries. DYNAMO1 converts ATP to GTP, and disruption of its activity impairs mitochondrial and peroxisomal fissions. DYNAMO1 forms a ring-shaped complex with Dnm1 and increases the magnitude of the constricting force. Our results identify DYNAMO1 as an essential component of MD and POD machineries, suggesting that local GTP generation in Dnm1-based machinery regulates motive force for membrane severance.

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

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.

Development of a Double Nuclear Gene-Targeting Method by Two-Step Transformation Based on a Newly Established Chloramphenicol-Selection System in the Red Alga Cyanidioschyzon merolae.

The unicellular red alga Cyanidioschyzon merolae possesses a simple cellular architecture that consists of one mitochondrion, one chloroplast, one peroxisome, one Golgi apparatus, and several lysosomes. The nuclear genome content is also simple, with very little genetic redundancy (16.5 Mbp, 4,775 genes). In addition, molecular genetic tools such as gene targeting and inducible gene expression systems have been recently developed. These cytological features and genetic tractability have facilitated various omics analyses. However, only a single transformation selection marker URA has been made available and thus the application of genetic modification has been limited. Here, we report the development of a nuclear targeting method by using chloramphenicol and the chloramphenicol acetyltransferase (CAT) gene. In addition, we found that at least 200-bp homologous arms are required and 500-bp arms are sufficient for a targeted single-copy insertion of the CAT selection marker into the nuclear genome. By means of a combination of the URA and CAT transformation systems, we succeeded in producing a C. merolae strain that expresses HA-cyclin 1 and FLAG-CDKA from the chromosomal CYC1 and CDKA loci, respectively. These methods of multiple nuclear targeting will facilitate genetic manipulation of C. merolae.

Stable expression of a GFP-reporter gene in the red alga Cyanidioschyzon merolae.

The unicellular red alga Cyanidioschyzon merolae is used as a model organism to investigate the basic architecture of photosynthetic eukaryotes. We established a stable expression system for the green fluorescent protein fused with the phycocyanin-associated rod linker (APCC) protein in C. merolae, which was clearly localized on the plastid. This system should be useful in the genetic engineering of C. merolae.

Gene targeting in the red alga Cyanidioschyzon merolae: single- and multi-copy insertion using authentic and chimeric selection markers.

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, URA Cm-Gs , has been available that complements the uracil-auxotrophic mutant M4. URA Cm-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 URA Cm-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 (URA Cm-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.

Identification of highly-disrupted tRNA genes in nuclear genome of the red alga, Cyanidioschyzon merolae 10D.

The limited locations of tRNA introns are crucial for eukaryal tRNA-splicing endonuclease recognition. However, our analysis of the nuclear genome of an early-diverged red alga, Cyanidioschyzon merolae, demonstrated the first evidence of nuclear-encoded tRNA genes that contain ectopic and/or multiple introns. Some genes exhibited both intronic and permuted structures in which the 3'-half of the tRNA coding sequence lies upstream of the 5'-half, and an intron is inserted into either half. These highly disrupted tRNA genes, which account for 63% of all nuclear tRNA genes, are expressed via the orderly and sequential processing of bulge-helix-bulge (BHB) motifs at intron-exon junctions and termini of permuted tRNA precursors, probably by a C. merolae tRNA-splicing endonuclease with an unidentified subunit architecture. The results revealed a considerable diversity in eukaryal tRNA intron properties and endonuclease architectures, which will help to elucidate the acquisition mechanism of the BHB-mediated disrupted tRNA genes.

Mitochondrial localization of ferrochelatase in a red alga Cyanidioschyzon merolae.

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.

Single-membrane-bounded peroxisome division revealed by isolation of dynamin-based machinery.

Peroxisomes (microbodies) are ubiquitous single-membrane-bounded organelles and fulfill essential roles in the cellular metabolism. They are found in virtually all eukaryotic cells and basically multiply by division. However, the mechanochemical machinery involved in peroxisome division remains elusive. Here, we first identified the peroxisome-dividing (POD) machinery. We isolated the POD machinery from Cyanidioschyzon merolae, a unicellular red alga containing a single peroxisome. Peroxisomal division in C. merolae can be highly synchronized by light/dark cycles and the microtubule-disrupting agent oryzalin. By proteomic analysis based on the complete genome sequence of C. merolae, we identified a dynamin-related protein 3 (DRP3) ortholog, CmDnm1 (Dnm1), that predominantly accumulated with catalase in the dividing-peroxisome fraction. Immunofluorescence microscopy demonstrated that Dnm1 formed a ring at the division site of the peroxisome. The outlines of the isolated dynamin rings were dimly observed by phase-contrast microscopy and clearly stained for Dnm1. Electron microscopy revealed that the POD machinery was formed at the cytoplasmic side of the equator. Immunoelectron microscopy showed that the POD machinery consisted of an outer dynamin-based ring and an inner filamentous ring. Down-regulation of Dnm1 impaired peroxisomal division. Surprisingly, the same Dnm1 serially controlled peroxisomal division after mitochondrial division. Because genetic deficiencies of Dnm1 orthologs in multiperoxisomal organisms inhibited both mitochondrial and peroxisomal proliferation, it is thought that peroxisomal division by contraction of a dynamin-based machinery is universal among eukaryotes. These findings are useful for understanding the fundamental systems in eukaryotic cells.

The kinesin-like protein TOP promotes Aurora localisation and induces mitochondrial, chloroplast and nuclear division.

The cell cycle usually refers to the mitotic cycle, but the cell-division cycle in the plant kingdom consists of not only nuclear but also mitochondrial and chloroplast division cycle. However, an integrated control system that initiates division of the three organelles has not been found. We report that a novel C-terminal kinesin-like protein, three-organelle division-inducing protein (TOP), controls nuclear, mitochondrial and chloroplast divisions in the red alga Cyanidioschyzon merolae. A proteomics study revealed that TOP is a member of a complex of mitochondrial-dividing (MD) and plastid-dividing (PD) machineries (MD/PD machinery complex) just prior to constriction. After TOP localizes at the MD/PD machinery complex, mitochondrial and chloroplast divisions occur and the components of the MD/PD machinery complexes are phosphorylated. Furthermore, we found that TOP downregulation impaired both mitochondrial and chloroplast divisions. MD/PD machinery complexes were formed normally at each division site but they were neither phosphorylated nor constricted in these cells. Immunofluorescence signals of Aurora kinase (AUR) were localized around the MD machinery before constriction, whereas AUR was dispersed in the cytosol by TOP downregulation, suggesting that AUR is required for the constriction. Taken together our results suggest that TOP induces phosphorylation of MD/PD machinery components to accomplish mitochondrial and chloroplast divisions prior to nuclear division, by relocalization of AUR. In addition, given the presence of TOP homologs throughout the eukaryotes, and the involvement of TOP in mitochondrial and chloroplast division may illuminate the original function of C-terminal kinesin-like proteins.

Golgi inheritance in the primitive red alga, Cyanidioschyzon merolae.

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