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

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 FRB domain of human target of rapamycin (TOR) kinase or its derivatives to knock down target proteins. In the inducible system, rapamycin treatment induces the heterodimerization of the human FKBP12-rapamycin binding (FRB) domain fused to the target proteins with the human FK506-binding protein 12 (FKBP) fused to S-phase kinase associated protein 1 (SKP1) or Cullin 1 (CUL1), 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 (DRP5B) and E2 transcription factor (E2F), a regulator of the G1/S transition, within 2-3 hours 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.

The structure of PSI-LHCI from Cyanidium caldarium provides evolutionary insights into conservation and diversity of red-lineage LHCs.

Light-harvesting complexes (LHCs) are diversified among photosynthetic organisms, and the structure of the photosystem I-LHC (PSI-LHCI) supercomplex has been shown to be variable depending on the species of organisms. However, the structural and evolutionary correlations of red-lineage LHCs are unknown. Here, we determined a 1.92-Å resolution cryoelectron microscopic structure of a PSI-LHCI supercomplex isolated from the red alga Cyanidium caldarium RK-1 (NIES-2137), which is an important taxon in the Cyanidiophyceae. We subsequently investigated the correlations of PSI-LHCIs from different organisms through structural comparisons and phylogenetic analysis. The PSI-LHCI structure obtained shows five LHCI subunits surrounding a PSI-monomer core. The five LHCIs are composed of two Lhcr1s, two Lhcr2s, and one Lhcr3. Phylogenetic analysis of LHCs bound to PSI in the red-lineage algae showed clear orthology of LHCs between C. caldarium and Cyanidioschyzon merolae, whereas no orthologous relationships were found between C. caldarium Lhcr1-3 and LHCs in other red-lineage PSI-LHCI structures. These findings provide evolutionary insights into conservation and diversity of red-lineage LHCs associated with PSI.

Taming the perils of photosynthesis by eukaryotes: constraints on endosymbiotic evolution in aquatic ecosystems.

An ancestral eukaryote acquired photosynthesis by genetically integrating a cyanobacterial endosymbiont as the chloroplast. The chloroplast was then further integrated into many other eukaryotic lineages through secondary endosymbiotic events of unicellular eukaryotic algae. While photosynthesis enables autotrophy, it also generates reactive oxygen species that can cause oxidative stress. To mitigate the stress, photosynthetic eukaryotes employ various mechanisms, including regulating chloroplast light absorption and repairing or removing damaged chloroplasts by sensing light and photosynthetic status. Recent studies have shown that, besides algae and plants with innate chloroplasts, several lineages of numerous unicellular eukaryotes engage in acquired phototrophy by hosting algal endosymbionts or by transiently utilizing chloroplasts sequestrated from algal prey in aquatic ecosystems. In addition, it has become evident that unicellular organisms engaged in acquired phototrophy, as well as those that feed on algae, have also developed mechanisms to cope with photosynthetic oxidative stress. These mechanisms are limited but similar to those employed by algae and plants. Thus, there appear to be constraints on the evolution of those mechanisms, which likely began by incorporating photosynthetic cells before the establishment of chloroplasts by extending preexisting mechanisms to cope with oxidative stress originating from mitochondrial respiration and acquiring new mechanisms.

The Polycomb repressive complex 2 deposits H3K27me3 and represses transposable elements in a broad range of eukaryotes.

The mobility of transposable elements (TEs) contributes to evolution of genomes. Their uncontrolled activity causes genomic instability; therefore, expression of TEs is silenced by host genomes. TEs are marked with DNA and H3K9 methylation, which are associated with silencing in flowering plants, animals, and fungi. However, in distantly related groups of eukaryotes, TEs are marked by H3K27me3 deposited by the Polycomb repressive complex 2 (PRC2), an epigenetic mark associated with gene silencing in flowering plants and animals. The direct silencing of TEs by PRC2 has so far only been shown in one species of ciliates. To test if PRC2 silences TEs in a broader range of eukaryotes, we generated mutants with reduced PRC2 activity and analyzed the role of PRC2 in extant species along the lineage of Archaeplastida and in the diatom P. tricornutum. In this diatom and the red alga C. merolae, a greater proportion of TEs than genes were repressed by PRC2, whereas a greater proportion of genes than TEs were repressed by PRC2 in bryophytes. In flowering plants, TEs contained potential cis-elements recognized by transcription factors and associated with neighbor genes as transcriptional units repressed by PRC2. Thus, silencing of TEs by PRC2 is observed not only in Archaeplastida but also in diatoms and ciliates, suggesting that PRC2 deposited H3K27me3 to silence TEs in the last common ancestor of eukaryotes. We hypothesize that during the evolution of Archaeplastida, TE fragments marked with H3K27me3 were selected to shape transcriptional regulation, controlling networks of genes regulated by PRC2.

Horizontally Acquired Nitrate Reductase Realized Kleptoplastic Photoautotrophy of Rapaza viridis.

While photoautotrophic organisms utilize inorganic nitrogen as the nitrogen source, heterotrophic organisms utilize organic nitrogen and thus do not generally have an inorganic nitrogen assimilation pathway. Here, we focused on the nitrogen metabolism of Rapaza viridis, a unicellular eukaryote exhibiting kleptoplasty. Although belonging to the lineage of essentially heterotrophic flagellates, R. viridis exploits the photosynthetic products of the kleptoplasts and was therefore suspected to potentially utilize inorganic nitrogen. From the transcriptome data of R. viridis, we identified gene RvNaRL, which had sequence similarity to genes encoding nitrate reductases in plants. Phylogenetic analysis revealed that RvNaRL was acquired by a horizontal gene transfer event. To verify the function of the protein product RvNaRL, we established RNAi-mediated knock-down and CRISPR-Cas9-mediated knock-out experiments for the first time in R. viridis and applied them to this gene. The RvNaRL knock-down and knock-out cells exhibited significant growth only when ammonium was supplied. However, in contrast to the wild-type cells, no substantial growth was observed when nitrate was supplied. Such arrested growth in the absence of ammonium was attributed to impaired amino acid synthesis due to the deficiency of nitrogen supply from the nitrate assimilation pathway; this in turn resulted in the accumulation of excess photosynthetic products in the form of cytosolic polysaccharide grains, as observed. These results indicate that RvNaRL is certainly involved in nitrate assimilation by R. viridis. Thus, we inferred that R. viridis achieved its advanced kleptoplasty for photoautotrophy, owing to the acquisition of nitrate assimilation via horizontal gene transfer.

Life cycle and functional genomics of the unicellular red alga Galdieria for elucidating algal and plant evolution and industrial use.

Sexual reproduction is widespread in eukaryotes; however, only asexual reproduction has been observed in unicellular red algae, including Galdieria, which branched early in Archaeplastida. Galdieria possesses a small genome; it is polyextremophile, grows either photoautotrophically, mixotrophically, or heterotrophically, and is being developed as an industrial source of vitamins and pigments because of its high biomass productivity. Here, we show that Galdieria exhibits a sexual life cycle, alternating between cell-walled diploid and cell wall-less haploid, and that both phases can proliferate asexually. The haploid can move over surfaces and undergo self-diploidization or generate heterozygous diploids through mating. Further, we prepared the whole genome and a comparative transcriptome dataset between the diploid and haploid and developed genetic tools for the stable gene expression, gene disruption, and selectable marker recycling system using the cell wall-less haploid. The BELL/KNOX and MADS-box transcription factors, which function in haploid-to-diploid transition and development in plants, are specifically expressed in the haploid and diploid, respectively, and are involved in the haploid-to-diploid transition in Galdieria, providing information on the missing link of the sexual life cycle evolution in Archaeplastida. Four actin genes are differently involved in motility of the haploid and cytokinesis in the diploid, both of which are myosin independent and likely reflect ancestral roles of actin. We have also generated photosynthesis-deficient mutants, such as blue-colored cells, which were depleted in chlorophyll and carotenoids, for industrial pigment production. These features of Galdieria facilitate the understanding of the evolution of algae and plants and the industrial use of microalgae.

Genome evolution of a nonparasitic secondary heterotroph, the diatom Nitzschia putrida.

Secondary loss of photosynthesis is observed across almost all plastid-bearing branches of the eukaryotic tree of life. However, genome-based insights into the transition from a phototroph into a secondary heterotroph have so far only been revealed for parasitic species. Free-living organisms can yield unique insights into the evolutionary consequence of the loss of photosynthesis, as the parasitic lifestyle requires specific adaptations to host environments. Here, we report on the diploid genome of the free-living diatom Nitzschia putrida (35 Mbp), a nonphotosynthetic osmotroph whose photosynthetic relatives contribute ca. 40% of net oceanic primary production. Comparative analyses with photosynthetic diatoms and heterotrophic algae with parasitic lifestyle revealed that a combination of gene loss, the accumulation of genes involved in organic carbon degradation, a unique secretome, and the rapid divergence of conserved gene families involved in cell wall and extracellular metabolism appear to have facilitated the lifestyle of a free-living secondary heterotroph.

A cotransformation system of the unicellular red alga Cyanidioschyzon merolae with blasticidin S deaminase and chloramphenicol acetyltransferase selectable markers.

BACKGROUND: The unicellular red alga Cyanidioschyzon merolae exhibits a very simple cellular and genomic architecture. In addition, procedures for genetic modifications, such as gene targeting by homologous recombination and inducible/repressible gene expression, have been developed. However, only two markers for selecting transformants, uracil synthase (URA) and chloramphenicol acetyltransferase (CAT), are available in this alga. Therefore, manipulation of two or more different chromosomal loci in the same strain in C. merolae is limited. RESULTS: This study developed a nuclear targeting and transformant selection system using an antibiotics blasticidin S (BS) and the BS deaminase (BSD) selectable marker by homologous recombination in C. merolae. In addition, this study has succeeded in simultaneously modifying two different chromosomal loci by a single-step cotransformation based on the combination of BSD and CAT selectable markers. A C. merolae strain that expresses mitochondrion-targeted mSCARLET (with the BSD marker) and mVENUS (with the CAT marker) from different chromosomal loci was generated with this procedure. CONCLUSIONS: The newly developed BSD selectable marker enables an additional genetic modification to the already generated C. merolae transformants based on the URA or CAT system. Furthermore, the cotransformation system facilitates multiple genetic modifications. These methods and the simple nature of the C. merolae cellular and genomic architecture will facilitate studies on several phenomena common to photosynthetic eukaryotes.

Genome sequencing of the multicellular alga Astrephomene provides insights into convergent evolution of germ-soma differentiation.

Germ-soma differentiation evolved independently in many eukaryotic lineages and contributed to complex multicellular organizations. However, the molecular genetic bases of such convergent evolution remain unresolved. Two multicellular volvocine green algae, Volvox and Astrephomene, exhibit convergent evolution of germ-soma differentiation. The complete genome sequence is now available for Volvox, while genome information is scarce for Astrephomene. Here, we generated the de novo whole genome sequence of Astrephomene gubernaculifera and conducted RNA-seq analysis of isolated somatic and reproductive cells. In Volvox, tandem duplication and neofunctionalization of the ancestral transcription factor gene (RLS1/rlsD) might have led to the evolution of regA, the master regulator for Volvox germ-soma differentiation. However, our genome data demonstrated that Astrephomene has not undergone tandem duplication of the RLS1/rlsD homolog or acquisition of a regA-like gene. Our RNA-seq analysis revealed the downregulation of photosynthetic and anabolic gene expression in Astrephomene somatic cells, as in Volvox. Among genes with high expression in somatic cells of Astrephomene, we identified three genes encoding putative transcription factors, which may regulate somatic cell differentiation. Thus, the convergent evolution of germ-soma differentiation in the volvocine algae may have occurred by the acquisition of different regulatory circuits that generate a similar division of labor.

The Unicellular Red Alga Cyanidioschyzon merolae-The Simplest Model of a Photosynthetic Eukaryote.

Several species of unicellular eukaryotic algae exhibit relatively simple genomic and cellular architecture. Laboratory cultures of these algae grow faster than plants and often provide homogeneous cellular populations exposed to an almost equal environment. These characteristics are ideal for conducting experiments at the cellular and subcellular levels. Many microalgal lineages have recently become genetically tractable, which have started to evoke new streams of studies. Among such algae, the unicellular red alga Cyanidioschyzon merolae is the simplest organism; it possesses the minimum number of membranous organelles, only 4,775 protein-coding genes in the nucleus, and its cell cycle progression can be highly synchronized with the diel cycle. These properties facilitate diverse omics analyses of cellular proliferation and structural analyses of the intracellular relationship among organelles. C. merolae cells lack a rigid cell wall and are thus relatively easily disrupted, facilitating biochemical analyses. Multiple chromosomal loci can be edited by highly efficient homologous recombination. The procedures for the inducible/repressive expression of a transgene or an endogenous gene in the nucleus and for chloroplast genome modification have also been developed. Here, we summarize the features and experimental techniques of C. merolae and provide examples of studies using this alga. From these studies, it is clear that C. merolae-either alone or in comparative and combinatory studies with other photosynthetic organisms-can provide significant insights into the biology of photosynthetic eukaryotes.

Cell size for commitment to cell division and number of successive cell divisions in cyanidialean red algae.

Several eukaryotic cell lineages proliferate by multiple fission cell cycles, during which cells grow to manyfold of their original size, then undergo several rounds of cell division without intervening growth. A previous study on volvocine green algae, including both unicellular and multicellular (colonial) species, showed a correlation between the minimum number of successive cell divisions without intervening cellular growth, and the threshold cell size for commitment to the first round of successive cell divisions: two times the average newly born daughter cell volume for unicellular Chlamydomonas reinhardtii, four times for four-celled Tetrabaena socialis, in which each cell in the colony produces a daughter colony by two successive cell divisions, and eight times for the eight-celled Gonium pectorale, in which each cell produces a daughter colony by three successive cell divisions. To assess whether this phenomenon is also applicable to other lineages, we have characterized cyanidialean red algae, namely, Cyanidioschyzon merolae, which proliferates by binary fission, as well as Cyanidium caldarium and Galdieria sulphuraria, which form up to four and 32 daughter cells (autospores), respectively, in a mother cell before hatching out. The result shows that there is also a correlation between the number of successive cell divisions and the threshold cell size for cell division or the first round of the successive cell divisions. In both C. merolae and C. caldarium, the cell size checkpoint for cell division(s) exists in the G1-phase, as previously shown in volvocine green algae. When C. merolae cells were arrested in the G1-phase and abnormally enlarged by conditional depletion of CDKA, the cells underwent two or more successive cell divisions without intervening cellular growth after recovery of CDKA, similarly to C. caldarium and G. sulphuraria. These results suggest that the threshold size for cell division is a major factor in determining the number of successive cell divisions and that evolutionary changes in the mechanism of cell size monitoring resulted in a variation of multiple fission cell cycle in eukaryotic algae.

Holliday Junction Resolvase MOC1 Maintains Plastid and Mitochondrial Genome Integrity in Algae and Bryophytes.

When DNA double-strand breaks occur, four-stranded DNA structures called Holliday junctions (HJs) form during homologous recombination. Because HJs connect homologous DNA by a covalent link, resolution of HJ is crucial to terminate homologous recombination and segregate the pair of DNA molecules faithfully. We recently identified Monokaryotic Chloroplast1 (MOC1) as a plastid DNA HJ resolvase in algae and plants. Although Cruciform cutting endonuclease1 (CCE1) was identified as a mitochondrial DNA HJ resolvase in yeasts, homologs or other mitochondrial HJ resolvases have not been identified in other eukaryotes. Here, we demonstrate that MOC1 depletion in the green alga Chlamydomonas reinhardtii and the moss Physcomitrella patens induced ectopic recombination between short dispersed repeats in ptDNA. In addition, MOC1 depletion disorganized thylakoid membranes in plastids. In some land plant lineages, such as the moss P. patens, a liverwort and a fern, MOC1 dually targeted to plastids and mitochondria. Moreover, mitochondrial targeting of MOC1 was also predicted in charophyte algae and some land plant species. Besides causing instability of plastid DNA, MOC1 depletion in P. patens induced short dispersed repeat-mediated ectopic recombination in mitochondrial DNA and disorganized cristae in mitochondria. Similar phenotypes in plastids and mitochondria were previously observed in mutants of plastid-targeted (RECA2) and mitochondrion-targeted (RECA1) recombinases, respectively. These results suggest that MOC1 functions in the double-strand break repair in which a recombinase generates HJs and MOC1 resolves HJs in mitochondria of some lineages of algae and plants as well as in plastids in algae and plants.

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.

Changes in the transcriptome, ploidy, and optimal light intensity of a cryptomonad upon integration into a kleptoplastic dinoflagellate.

Endosymbiosis of unicellular eukaryotic algae into previously nonphotosynthetic eukaryotes has established chloroplasts in several eukaryotic lineages. In addition, certain unicellular organisms in several different lineages ingest algae and utilize them as temporal chloroplasts (kleptoplasts) for weeks to months before digesting them. Among these organisms, the dinoflagellate Nusuttodinium aeruginosum ingests the cryptomonad Chroomonas sp. and enlarges the kleptoplast with the aid of the cryptomonad nucleus. To understand how the cryptomonad nucleus is remodeled in the dinoflagellate, here we examined changes in the transcriptome and ploidy of the ingested nucleus. We show that, after ingestion, genes involved in metabolism, translation, and DNA replication are upregulated while those involved in sensory systems and cell motility are downregulated. In the dinoflagellate cell, the cryptomonad nucleus undergoes polyploidization that correlates with an increase in the mRNA levels of upregulated genes. In addition, the ingested nucleus almost loses transcriptional responses to light. Because polyploidization and loss of transcriptional regulation are also known to have occurred during the establishment of endosymbiotic organelles, these changes are probably a common trend in endosymbiotic evolution. Furthermore, we show that the kleptoplast and dinoflagellate are more susceptible to high light than the free-living cryptomonad but that the ingested nucleus reduces this damage.

Relationship between Cell Cycle and Diel Transcriptomic Changes in Metabolism in a Unicellular Red Alga.

Metabolism, cell cycle stages, and related transcriptomes in eukaryotic algae change with the diel cycle of light availability. In the unicellular red alga Cyanidioschyzon merolae, the S and M phases occur at night. To examine how diel transcriptomic changes in metabolic pathways are related to the cell cycle and to identify all genes for which mRNA levels change depending on the cell cycle, we examined diel transcriptomic changes in C. merolae In addition, we compared transcriptomic changes between the wild type and transgenic lines, in which the cell cycle was uncoupled from the diel cycle by the depletion of either cyclin-dependent kinase A or retinoblastoma-related protein. Of 4,775 nucleus-encoded genes, the mRNA levels of 1,979 genes exhibited diel transcriptomic changes in the wild type. Of these, the periodic expression patterns of 454 genes were abolished in the transgenic lines, suggesting that the expression of these genes is dependent on cell cycle progression. The periodic expression patterns of most metabolic genes, except those involved in starch degradation and de novo deoxyribonucleotide triphosphate synthesis, were not affected in the transgenic lines, indicating that the cell cycle and transcriptomic changes in most metabolic pathways are independent of the diel cycle. Approximately 40% of the cell-cycle-dependent genes were of unknown function, and approximately 19% of these genes of unknown function are shared with the green alga Chlamydomonas reinhardtii The data set presented in this study will facilitate further studies on the cell cycle and its relationship with metabolism in eukaryotic algae.

Evolutionary Changes in DnaA-Dependent Chromosomal Replication in Cyanobacteria.

Replication of the circular bacterial chromosome is initiated at a unique origin (oriC) in a DnaA-dependent manner in which replication proceeds bidirectionally from oriC to ter. The nucleotide compositions of most bacteria differ between the leading and lagging DNA strands. Thus, the chromosomal DNA sequence typically exhibits an asymmetric GC skew profile. Further, free-living bacteria without genomes encoding dnaA were unknown. Thus, a DnaA-oriC-dependent replication initiation mechanism may be essential for most bacteria. However, most cyanobacterial genomes exhibit irregular GC skew profiles. We previously found that the Synechococcus elongatus chromosome, which exhibits a regular GC skew profile, is replicated in a DnaA-oriC-dependent manner, whereas chromosomes of Synechocystis sp. PCC 6803 and Nostoc sp. PCC 7120, which exhibit an irregular GC skew profile, are replicated from multiple origins in a DnaA-independent manner. Here we investigate the variation in the mechanisms of cyanobacterial chromosome replication. We found that the genomes of certain free-living species do not encode dnaA and such species, including Cyanobacterium aponinum PCC 10605 and Geminocystis sp. NIES-3708, replicate their chromosomes from multiple origins. Synechococcus sp. PCC 7002, which is phylogenetically closely related to dnaA-lacking free-living species as well as to dnaA-encoding but DnaA-oriC-independent Synechocystis sp. PCC 6803, possesses dnaA. In Synechococcus sp. PCC 7002, dnaA was not essential and its chromosomes were replicated from a unique origin in a DnaA-oriC independent manner. Our results also suggest that loss of DnaA-oriC-dependency independently occurred multiple times during cyanobacterial evolution and raises a possibility that the loss of dnaA or loss of DnaA-oriC dependency correlated with an increase in ploidy level.

ESCRT Machinery Mediates Cytokinetic Abscission in the Unicellular Red Alga Cyanidioschyzon merolae.

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.

Responses of unicellular predators to cope with the phototoxicity of photosynthetic prey.

Feeding on unicellular photosynthetic organisms by unicellular eukaryotes is the base of the aquatic food chain and evolutionarily led to the establishment of photosynthetic endosymbionts/organelles. Photosynthesis generates reactive oxygen species and damages cells; thus, photosynthetic organisms possess several mechanisms to cope with the stress. Here, we demonstrate that photosynthetic prey also exposes unicellular amoebozoan and excavates predators to photosynthetic oxidative stress. Upon illumination, there is a commonality in transcriptomic changes among evolutionarily distant organisms feeding on photosynthetic prey. One of the genes commonly upregulated is a horizontally transferred homolog of algal and plant genes for chlorophyll degradation/detoxification. In addition, the predators reduce their phagocytic uptake while accelerating digestion of photosynthetic prey upon illumination, reducing the number of photosynthetic cells inside the predator cells, as this also occurs in facultative endosymbiotic associations upon certain stresses. Thus, some mechanisms in predators observed here probably have been necessary for evolution of endosymbiotic associations.

Dynamin-Like Protein B of Dictyostelium Contributes to Cytokinesis Cooperatively with Other Dynamins.

Dynamin is a large GTPase responsible for diverse cellular processes, such as endocytosis, division of organelles, and cytokinesis. The social amoebozoan, Dictyostelium discoideum, has five dynamin-like proteins: dymA, dymB, dlpA, dlpB, and dlpC. DymA, dlpA, or dlpB-deficient cells exhibited defects in cytokinesis. DlpA and dlpB were found to colocalize at cleavage furrows from the early phase, and dymA localized at the intercellular bridge connecting the two daughter cells, indicating that these dynamins contribute to cytokinesis at distinct dividing stages. Total internal reflection fluorescence microscopy revealed that dlpA and dlpB colocalized at individual dots at the furrow cortex. However, dlpA and dlpB did not colocalize with clathrin, suggesting that they are not involved in clathrin-mediated endocytosis. The fact that dlpA did not localize at the furrow in dlpB null cells and vice versa, as well as other several lines of evidence, suggests that hetero-oligomerization of dlpA and dlpB is required for them to bind to the furrow. The hetero-oligomers directly or indirectly associate with actin filaments, stabilizing them in the contractile rings. Interestingly, dlpA, but not dlpB, accumulated at the phagocytic cups independently of dlpB. Our results suggest that the hetero-oligomers of dlpA and dlpB contribute to cytokinesis cooperatively with dymA.