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

Abstract

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 Sulfolobus. 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 Cyanidioschyzon merolae 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.

It is thought that mitochondria and plastids arose about 1–2 billion years ago when autonomous α-proteobacteria and cyanobacteria lived together in the cytoplasm of a eukaryotic ancestor, which resembles a thermophile archaeon Sulforobus in a form of cell division (Yagisawa et al. 2020) and had emerged from Asgard archaea (Zaremba-Niedzwiedzka et al. 2017). The symbiotic α-proteobacteria lost autonomy and were integrated with the host organism via endosymbiotic gene transfer and converted to mitochondria. Remaining copies of the mtDNA were then duplicated in the primitive unicellular organisms in response to cell function.

Today, there appear to be three types of mt-nuclei in eukaryotes. The first type is unicellular organisms with one mitochondrion containing one mt-nucleus including many copies (10–100) of the mtDNA. Examples are microalgae such as C. merolae (Matsuzaki et al. 2004), Ostereococcus tauri, and Medakamo hakoo (Kuroiwa et al. 2004, 2016). The second type are unicellular organisms with multiple mitochondria containing some mt-nuclei that can be fused into a few huge threads of fused reticulum surrounding the cell nucleus, for example, Saccharomyces cerevisiae (Sando et al. 1981, Miyakawa et al. 1984, 1987, 1991). The third type includes many Plantae, Amoebozoa, Fungi, and Animals. The cells of these organisms contain many mitochondria with some mt-nuclei because the events of mitochondrial division and mtDNA synthesis are not synchronized (Nishibayashi and Kuroiwa 1985).

Multiple copies of mtDNA are organized by DNA binding proteins in various organisms including yeast (Miyakawa et al. 1987, Miyakawa 2017), slime molds (Sasaki et al. 1998, 2003, Itoh et al. 2009, 2011), algae, and plants (Sakai et al. 2004, Kobayashi et al. 2017), and animals (Satoh and Kuroiwa 1991). However, despite extensive research toward the genetic or proteomic identification of the DNA binding proteins, such as Grom and HU-like proteins (Sasaki et al. 1998, 2003, Kobayashi et al. 2002, Itoh et al. 2009, 2011), it is not clear how mtDNA copies are organized morphologically. The red alga C. merolae diverged early in eukaryotic evolution (Frederick and Seckbach 1986). C. merolae cells are advantageous for studying mt-nuclear dynamics because the cell contains only one cell nucleus, one mitochondrion, and one plastid of the simplest morphology (Fig. 1Aa), whose division can be easily and highly synchronized using light and dark cycles (Suzuki et al. 1994, Terui et al 1995, Imoto et al. 2010, 2011, 2018); their genomes are 16.5 Mb, 35 kb and 150 kb, respectively and 100% sequenced (Ohta et al. 1998, Matsuzaki et al. 2004, Nozaki et al. 2007). This species is amenable to many genetic techniques, as well as genome-wide and system-wide multiomics analyses (Fujiwara et al. 2013). Furthermore, the cell wall is not rigid, and thus isolation of intracellular organelles is possible, as reported in many studies (Yoshida et al. 2006, 2010, 2017, Kuroiwa et al. 2017, Yagisawa et al. 2017).

Fig. 1. Model and DAPI-stained fluorescent micrograph of the unicellular red alga C. merolae cell without and with the application of the drying method used in this study. (A) Cell model indicating the cell nucleus, mt-nucleus, and pt- nucleus (a) and a fluorescence micrograph of the cell after staining with DAPI without the drying treatment (b). (B) Fluorescence micrographs of cells after staining with DAPI using the drying treatment. The centrally localized pt-nuclei changed into ring-shaped pt-nuclei (short arrow in a). Small units of pt-nuclear rings appeared in the swollen pt-nucleus (short arrow in b), but the mt-nucleus was unclear (long arrow in b). n, cell nucleus; m, mitochondrial nucleus; p, plastid nucleus. Scale bar=1 µm.

Recently a new technique to observe plastid nuclei, and mitotic chromosomes in C. merolae was developed. When C. merolae cells were stained with SYBR Green after drying treatment, a bead combination ring-like pt-nucleus (Fig. 1d) (Kuroiwa et al. 2020a) and mitotic chromosomes (Kuroiwa et al. 2020b) became visible. However, the mt-nuclei were not clear. Here, using an improved drying treatment, we observed the mt-nucleus. The mt-nucleus, which was centrally located in the mitochondrion, could be changed to a smooth ring-like structure. The observed images appear to be the primary structure of organized mitochondrial DNA. Finally, the evolutionary significance of the smooth ring-like mt-nuclei and the bead combination ring-like pt-nuclei are discussed.

Materials and methods

Cell culture

In this study, we used C. merolae strain 10D, which was established by Toda et al. (1995), cultured in Misumi–Kuroiwa (MK) medium at pH 1.8 and 42°C. The MK medium was prepared by diluting 1 mL of a commercial nutrient solution (Hyponex, N : P : K 10 : 8 : 8; Hyponex Japan, Osaka, Japan) to 1 L with distilled or tap water. Both media were adjusted to pH 1.8 with 1 mL concentrated hydrochloric acid (Kuroiwa et al. 2020a, b). Cells were maintained under continuous light (60 µmol m⁻² s⁻¹). The mitotic index was about 12%.

4′6-Diamidino-2-phenyindole (DAPI) and SYBR Green I staining

Conventional DAPI staining (Kuroiwa and Suzuki 1980) was performed to confirm the bead combination ring-like pt-nuclei shown by SYBR Green (Molecular Probes, Eugene, OR, USA) staining (Kuroiwa et al. 2020a, b). Then, to avoid the influence of G+C content on the stainability of DNA, cells were stained with SYBR Green I, which has been used to stain pt-nuclei in various algae (Nishimura et al. 1998). After resuspending a pellet of C. merolae obtained from centrifuged culture, a 3-µL aliquot of the solution was placed on a slide glass. Next, 3 µL of 1% (v/v) glutaraldehyde buffered with NS buffer (Nishibayashi et al. 1987) was added to the drop, followed by 3 µL DAPI (15 µg mL⁻¹) or 3 µL SYBR Green 1 (1 µg mL⁻¹). After the coverslip was put on the mixed drop, and squashed with weak pressure. The samples were observed using a fluorescence microscope.

For the drying treatment, a 3-µL aliquot of C. merolae culture medium and 3 µL of 0.2–0.5% glutaraldehyde were placed on a glass slide and air-dried. The slide was tilted during air-drying and 2 µL DAPI or 3 µL SYBR Green 1 was placed on the dried samples. After the coverslip was put on the mixed drop, and squashed with weak pressure, another 1 µL DAPI or 2 µL SYBR Green 1 was added to the edge of the coverslip. The samples were observed using a fluorescence microscope.

Fluorescence microscopy and measurement of fluorescence intensity using a video-intensified microscope photon-counting system (VIMPCS)

The stained samples were observed using an Olympus BH-2 BHS epifluorescence microscope. The fluorescence intensities of the smooth ring-like mt-nuclei were measured using a VIMPCS (Miyamura et al. 1986). This basic method was used previously to analyze a ring-shaped-pt-nucleus in a single petal-like chloroplast of the red alga Galdieria sulphuraria (Kuroiwa et al. 1989).

Results

In C. merolae cell, one cell nucleus, one mitochondrion, and one chloroplast line up (Fig. 1Aa). When C. merolae cells were stained with DAPI without the drying treatment, these three organelles showed each organelle nucleus (Fig. 1Ab). The pt-nucleus was present as a spherule in the central area of the plastid (Fig. 1Ab). By contrast, when C. merolae cells were stained with DAPI after the drying treatment, bead combination ring-like pt-nuclei appeared at the periphery of the plastid (Fig. 1Ba). As the plastid swelled further, bead units with a diameter of about 0.2 µm became clear, and the beads appeared to be partially connected (Fig. 1Bb).

When C. merolae cells were stained with SYBR Green 1 after the drying treatment, a smooth loop-like mt-nucleus appeared next to the cell nucleus (Fig. 2A). Although mitochondria were present between the cell nucleus and the plastid before the drying treatment (Fig. 1A), mt-nuclei moved away from broken mitochondria and to the opposite side of the pt-nucleus after the drying treatment (long arrows in Fig. 2A). In swelled plastids, bead-like units of the pt-nuclei could be visualized (short arrows in Fig. 2A). Typical mt-nuclei were observed opposite of the pt-nuclei (Fig. 2Ba, b).

Fig. 2. Fluorescent micrographs showing cell nuclei, mt-nuclei and pt-nuclei in several cells after staining with SYBR Green 1 using the drying treatment. (A) One mt-nucleus, one cell nucleus, and one pt-nucleus are aligned in most cells. Mt-nuclei moved in the cytosol to the opposite side of the pt-nucleus (long arrows). Small units of pt-nuclear rings appeared in swollen pt-nuclei (short arrows). (B) Two typical types of cells were observed. In one, the mt-nucleus was next to the cell nucleus (a); in the other, the mt-nucleus was located opposite to the pt-nucleus (b). The mt-nuclear ring was smooth and had a structure that contrasts with the pt-nuclear ring, which looks like a series of bead-like small units. m, mt-nuclear ring; n, cell nucleus; p, pt-nuclear ring; PC, phase contrast; SYG, SYBR Green I; PC+SYG, combined PC and SYG; AUT, autofluorescence of chloroplast chlorophyll. Scale bar=1 µm.

The mt-nucleus had a smooth loop structure and associated with the cell nucleus (Figs. 2–4). When the pt-nuclei were contracted in some cells, the length of the mt-nuclei was variable, suggesting that a weak force was applied from the pt-nucleus side (Fig. 3a–e). Smooth, closed mt-nuclear rings were observed in some cells (Fig. 3b, c). The circumference of the closed rings was about 3.9 µm, close to the average length of the protruding mt-nuclei (Table 1). Long, folded mt-nuclear rings and oval cell nuclei, which would be caused by the hydraulic force during drying, were observed (Fig. 4a–d). It should be noted that the mt-nuclei were smooth (Fig. 4A). When the plastid was removed from the cell but part of the bead combination ring-like pt-nucleus remained, the difference between the smooth mt-nucleus and the pt-nucleus became clear (Fig. 4B).

Fig. 3. Fluorescence micrographs of cells in which a weak force was applied from the pt-nuclear side and the pt-nuclei were compressed when SYBR Green 1 was added to the edge of the coverslip. The mt-nuclei were swollen and their ring structures were clarified. The mt-nuclei were swollen and the mt-nuclear rings were smooth (a–e). In some cases, the mt-nuclei elongated slightly from the cell nuclei and formed a complete ring (arrows in b, c). In addition, double mt-nuclear rings that seem to be daughter mt-nuclei were seen (arrow in f). m, mt-nucleus; n, cell nucleus; p, pt-nucleus; PC, phase contrast; SYG, SYBR Green 1; PC+SYG, combined PC and SYG; AUT, autofluorescence of chloroplast chlorophyll. Scale bar=1 µm.

Fig. 4. Fluorescence micrographs of cells in which a weak force was applied from one side of the cell and the mt-nuclei and cell nuclei were elongated. (A). A long, smooth mt-nuclear loop extending out from the oval cell nuclei was visible (a–d). Especially long loops reached about 4 µm (total 8 µm) (arrow in c). (B) In the plastid-detached cell, a fragment of pt-nucleus with small bead-like units was attached to the cell nucleus and was different from the smooth mt-nuclear ring. m, mt-nucleus; n, cell nucleus; p, pt-nucleus; PC, phase contrast; SYG, SYBR Green I; PC+SYG, combined PC and SYG; AUT, autofluorescence of chloroplast chlorophyll. Scale bar=1 µm.

When the amount of DNA in the closed mt-nuclear rings was measured by VIMPCS, it was 2.9 Mb on average (Table 1). Because the size of the C. merolae mitochondrial genome is 0.035 Mb, it is estimated that one loop-shaped mt-nucleus is composed of 83 mtDNA copies. The mtDNA copy number per mt-nucleus is similar to that of ptDNA in the pt-nucleus (Table 1).

Table 1. Structure and size of the genome, and mitochondrial nucleus, and plastid nucleus of C. merolae.

	Genome	Organelle nucleus (nucleoid)
	Structure Size (Mb)	Structure (length µm)	DNA content (Mb)	Copy number
Mitochondrial nucleus	circle 0.035	smooth ring (3.8±1.4)	2.9±0.9	83
Cell nucleus	16.5
Plastid nucleus	circle 0.15	beads bound-like ring (4.3±0.9)	12.0±1.7	80

Mean±SD. Typical mt-nuclei and pt-nuclei during G₁ phase were examined. Data are mean photon counts from organelle nucleus per 0.1 s after SYBR Green I staining. Genome sizes of the cell nucleus and the plastid were obtained from Matsuzaki et al. (2004) and Ohta et al. (2003), respectively

Our study does not provide data on how the smooth ring-like mt-nuclei divide; this is a topic that should be addressed in the future. But at this stage, we can speculate a little. A completely closed mt-nucleus with a circumference of about 3.9 µm was observed (Fig. 3a, c). When the cells were squashed in one direction by a weak force, the mt-nuclei were elongated, the loops overlapped, and their length halved (Fig. 4A); these results suggest that the mt-nuclei elongated to about 8 µm and split in two. The appearance of double smooth ring-like mt-nuclei may be consistent with this idea (Fig. 3f).

Discussion

It is thought that mitochondria and plastids arose about 1–2 billion years ago when autonomous α-proteobacteria and cyanobacteria lived together in the cytoplasm of a host organism. Following many studies about the symbiotic phenomena, it became possible to understand the conversion of symbiotic bacteria into mitochondria and plastids after the symbiosis. However, there are few studies on the origin and evolution of the host organism. Recently, it has been demonstrated that the endosomal sorting complex required for transport III (ESCRT-III) and vacuolar protein sorting 4 (Vps4) mediate the abscission of cytokines from the archaeon Sulfolobus to almost every eukaryote, including Plantae, Amoebozoa, Fungi, and Animals (Yagisawa et al. 2020). This suggests that the host organism was a Sulfolobus-like species. A crenarchaeal genus, Sulfolobus lives in thermoacidophilic environments (Whitaker et al. 2005). This is not in conflict with the idea that the original eukaryotes may be Cyanidiophyceae such as G. sulphuraria, Cyanidium caldarium and C. merolae, which also live in thermoacidophilic environments (Nagashima et al. 1984, Seckbach 1995).

When C. merolae cells were stained with SYBR Green 1, circular pt-nuclei composed of small pt-nuclear units were observed (Kuroiwa et al. 2020a). In the present study, when the plastid swelled slightly, 0.4-µm diameter pt-nuclear units were clearly visible after DAPI staining and they seemed to be partly connected (Fig. 1Bb). Similar bead combination ring-like pt-nuclei are seen in red algae such as G. sulphuraria (Cyanidium caldarium M8) of the Rhodophyta lineage, and in brown algae of the Heterokontophyta lineage such as Ectocarpus siliculosus, Licmophora abbreviata, and Nitzschia sp. (Kuroiwa et al. 1981, 1982, 1989, Misumi et al. 2001). In the green alga Chlamydomonas reinhardtii and higher plants, bead like pt-nuclei are scattered throughout plastids (Sakai et al. 2004, Kobayashi et al. 2017).

The mt-nucleus of C. merolae formed a smooth-shaped loop and contained approximately 83 mtDNA copies (Table 1). Even when the mt-nucleus was stretched, bead-like granules did not appear on the ring (Fig. 4). In the yeast S. cerevisiae, one mesh-like mitochondrion is formed by the fusion of many small mitochondria each with a small number of copies of the mtDNA. Although the large mesh-like mt-nucleus contains many copies of the mtDNA, it is smooth (Miyakawa et al. 1987, 1991, Miyakawa 2017). Smooth mt-nuclei were observed in Toxoplasma gondii (Matsuzaki et al. 2001). In Physarum polycephalum, multiple mitochondria have an electron-dense rod-shaped mt-nucleus which contains 32 genome copies during the G₁ stage of the mitochondrial division cycle (Kuroiwa 1982, Kawano et al. 1983). Although the DNA content per mitochondrion decreased remarkably with spherulation and the mt-nucleus became short, the mt-nuclei were smooth. Similar images were recorded for isolated mt-nuclei of P. polycephalum (Sasaki et al. 1998, 2003, Itoh et al. 2009, 2011). On the basis of these results, we suggest that mtDNA molecules assemble smooth loop-shaped mitochondrial nuclei by a mechanism that is conserved in various eukaryotes.

In most eukaryotes, mtDNA and ptDNA are generally circular DNAs (Ohta et al. 1993, 2003, Gillham 1994, Alberts et al. 2002). Nevertheless, the structure of the mt-nucleus differs from that of the pt-nucleus (Fig. 5).

Fig. 5. Model of the unicellular red alga C. merolae cell without and after application of the drying method. (A) A traditional cell model showing the cell nucleus (n), mitochondrial nucleus (m) and plastid nucleus (p) without the drying treatment. (B) New cell and organelle nuclear model showing mitotic chromosomes (Kuroiwa et al. 2020), smooth ring-shaped mt-nucleus, and bead combination ring-like pt-nucleus following the drying treatment.

With the evolution of cell function, the number of mitochondria in the cell increased to several hundred in amoeba and multicellular organisms. Additionally, the mitochondria become small, and the mtDNA copy number was reduced. In these organisms, mt-nuclear division has been observed, but the molecular mechanism is unknown (Nishibayashi et al. 1987). In the present study, when the mt-nuclear loop was elongated, double mt-loops were occasionally observed (Fig. 3f). Circular type pt-nuclear division was observed in the brown alga E. siliculosus (Kuroiwa and Suzuki 1981). The molecular mechanism of division of the smooth loop-like mt-nuclear may be similar to that of ring-shaped pt-nuclei.

It would be interesting to know how many mt-DNA molecules organize into a single smooth loop-shaped mt-nucleus, and how it divides evenly. Similarly, the organization of pt-DNA molecules in the pt-nucleus and pt-nuclear division are interesting (Misumi et al. 1999). Elucidation of these processes at the molecular level remains a challenge for the future.

Acknowledgments

This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (16H0483 and 19H03260) to T. K. We thank Edanz Group (https://en-author-services.edanzgroup.com/ac) for editing a draft of this manuscript.

References

• Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K. and Walter, P. 2002. Molecular Biology of the Cell. Garland Sciences, New York.
• Frederick, J. K. and Seckbach, J. 1986. Storage glucan and glucosyltransferase isozymes of Cyanidoschyzon merolae: A primitive eukaryote. Phytochemistry 25: 363–366.
• Fujiwara, T., Tanaka, K., Kuroiwa, T. and Hirano, T. 2013. Spatiotemporal dynamics of Condensins I and II: Evolutionary insights from the primitive red alga Cyanidioschyzon merolae. Mol. Biol. Cell 24: 2515–2527.
• Gillham, N. W. 1994. Organelle Genes and Genomes. Oxford University Press, New York.
• Imoto, Y., Abe, Y., Honsho, M., Okumoto, K., Ohnuma, M., Kuroiwa, H., Kuroiwa, T. and Fujiki, Y. 2018. Onsite GTP fuelling via DYNAMO1 drives division of mitochondria and peroxisomes. Nat. Commun. 9: 4634.
• Imoto, Y., Fujiwara, T., Yoshida, Y., Kuroiwa, H., Maruyama, S. and Kuroiwa, T. 2010. Division of cell nuclei, mitochondria, plastids and microbodies mediated by mitotic spindle poles in the primitive red alga Cyanidioschyzon merolae. Protoplasma 241: 64–74.
• Imoto, Y., Yoshida, Y., Yagisawa, F., Kuroiwa, H. and Kuroiwa, T. 2011. The cell cycle, including the mitotic cycle and organelle division cycle, as revealed by cytological observations. J. Electron Microsc. 60 Suppl 1: S117–S136.
• Itoh, K., Izumi, A., Mori, T., Dohmae, N., Yui, R., Maeda-Sano, K., Kanaoka, M. M., Kuroiwa, H., Kuroiwa, T., Higashiyama, T., Murakami-Murofushi, K., Kawano, S. and Sasaki, N. 2009. New protein Pmn34 with an exonuclease motif localizes in the mitochondrial nucleoid periphery of Physarum polycephalum. Cytologia 74: 401–407.
• Itoh, K., Izumi, A., Mori, T., Dohmae, N., Yui, R., Maeda-Sano, K., Shirai, Y., Kanaoka, M. M., Kuroiwa, T., Higashiyama, T., Sugita, M., Murakami-Murofushi, K., Kawano, S. and Sasaki, N. 2011. DNA packaging proteins Glom and Glom2 coordinately organize the mitochondrial nucleoid of Physarum polycephalum. Mitochondrion 11: 575–586.
• Kawano, S., Nishibayashi, S., Shiraishi, N., Miyahara, M. and Kuroiwa, T. 1983. Variance of ploidy in mitochondrial nucleus during spherulation in Physarum polycephalum. Exp. Cell Res. 149: 359–373.
• Kobayashi, Y., Misumi, O., Odawara, M., Ishibashi, K., Hirono, M., Hidaka, K., Endo, M., Sugiyama, H., Iwasaki, H., Kuroiwa, T., Shikanai, T. and Nishimura, Y. 2017. Holliday junction resolvases mediate chloroplast nucleoid segregation. Science 356: 631–634.
• Kobayashi, T., Takahara, M., Miyagishima, S., Kuroiwa, H., Sasaki, N., Ohta, N. and Kuroiwa, T. 2002. Detection and localization of a chloroplast-encoded HU-like protein that may organize chloroplast nucleoids. Plant Cell 14: 1579–1589.
• Kuroiwa, T. 1982. Mitochondrial nuclei. Int. Rev. Cytol. 75: 1–59.
• Kuroiwa, T., Kawano, S., Nishibayashi, S. and Sato, C. 1982. Epifluorescent microscopic evidence for maternal inheritance of chloroplast DNA. Nature 298: 481–483.
• Kuroiwa, T., Kuroiwa, T., Nozaki, H., Matsuzaki, M., Misumi, O. and Kuroiwa, H. 2004. Does cell size depend on the nuclear genome size in ultra-small algae such as Cyanidioschyzon merolae and Ostreococcus tauri? Cytologia 69: 93–96.
• Kuroiwa, T., Miyagishima, S., Matsunaga, S., Sato, N., Nozaki, H., Tanaka, K. and Misumi, O. (eds.) 2017. Cyanidioschyzon merolae: A New Model Eukaryote for Cell and Organelle Biology. Springer Nature, Singapore.
• Kuroiwa, T., Nagashima, H. and Fukuda, I. 1989. Chloroplast division without DNA synthesis during the life cycle of the unicellular alga Cyanidium caldarium M-8 as revealed by quantitative fluorescence microscopy. Protoplasma 149: 120–129.
• Kuroiwa, T., Ohnuma, M., Imoto, Y., Misumi, O., Nagata, N., Miyakawa, I., Fujishima, M. and Kuroiwa, H. 2016. Cell-nuclear genome size of the ultrasmall unicellular freshwater green alga, Medakamo hakoo 311 as studied by DAPI staining after microwave oven treatments: II. Comparison with Cyanidioschyzon merolae, Saccharomyces cerevisiae (n, 2n) and Chlorella variabilis. Cytologia 81: 69–76.
• Kuroiwa, T., Ohnuma, M., Imoto, Y., Yagisawa, Y., Misumi, O., Nagata, N. and Kuroiwa, H. 2020a. Evolutionary significance of the ring-like plastid nucleus in the primitive red alga Cyanidioschyzon merolae as revealed by drying. Protoplasma 257: 1069–1078.
• Kuroiwa, T. and Suzuki, T. 1980. An improved method for the demonstration of in situ chloroplast nuclei in higher plants. Cell Struct. Funct. 5: 195–197.
• Kuroiwa, T. and Suzuki, T. 1981. Circular nucleoids from chloroplasts in a brown alga Ectocarpus siliculosus. Exp. Cell Res. 134: 457–461.
• Kuroiwa, T., Suzuki, T., Ogawa, K. and Kawano, S. 1981. The chloroplast nucleus distribution, number, size and shape, and a model for the multiplication of the chloroplast genome during chloroplast development. Plant Cell Physiol. 22: 381–396.
• Kuroiwa, T., Yagisawa, F., Fujiwara, T., Inui, Y., Matsunaga, M. T., Kato, S., Matsunaga, S., Nagata, N., Imoto, Y. and Kuroiwa, H. 2020b. Mitotic karyotype of the primitive red alga Cyanidioschyzon merolae 10D. Cytologia 85: 1–7.
• Matsuzaki, M., Kikuchi, T., Kita, K., Kojima, S. and Kuroiwa, T. 2001. Large amounts of apicoplast nucleoid DNA and its segregation in Toxoplasma gondii. Protoplasma 218: 180–191.
• Matsuzaki, M. et al. 2004. Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D. Nature 428: 653–657.
• Misumi, O., Nishimura, Y. and Kuroiwa, T. 2001. Effects of chloroplast DNA content on the cell proliferation and aging in Chlamydomonas reinhardtii. J. Plant Res. 114: 125–131.
• Misumi, O., Suzuki, L., Nishimura, Y., Sakai, A., Kawano, S., Kuroiwa, H. and Kuroiwa, T. 1999. Isolation and phenotypic characterization of Chlamydomonas reinhardtii mutants defective in chloroplast DNA segregation. Protoplasma 209: 273–282.
• Miyakawa, I. 2017. Organization and dynamics of yeast mitochondrial nucleoids. Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. 93: 339–359.
• Miyakawa, I., Aoi, H., Sando, N. and Kuroiwa, T. 1984. Fluorescence microscopic studies of mitochondrial nucleoids during meiosis and sporulation in the yeast, Saccharomyces cerevisiae. J. Cell Sci. 66: 21–38.
• Miyakawa, I., Sando, N., Kawano, S., Nakamura, S. and Kuroiwa, T. 1987. Isolation of morphologically intact mitochondrial nucleoids from the yeast, Saccharomyces cerevisiae. J. Cell Sci. 88: 431–439.
• Miyakawa, I., Sando, N. and Kuroiwa, T. 1991. Nature 349: Cover, v.
• Miyamura, S., Nagata, T. and Kuroiwa, T. 1986. Quantitative fluorescence microscopy on dynamic changes of plastid nucleoids during wheat development. Protoplasma 133: 66–72.
• Nagashima, H., Kuroiwa, T. and Fukuda, I. 1984. Chloroplast nucleoids in a unicellular hot spring alga Cyanidium caldarium and related algae. Experientia 40: 563–564.
• Nishibayashi, S., Kawano, S. and Kuroiwa, T. 1987. Light and electron microscopic observations of mitochondrial fusion in plasmodia induced sporulation in Physarum polycephalum. Cytologia 52: 599–614.
• Nishibayashi, S. and Kuroiwa, T. 1985. Division of mitochondrial nuclei in protozoa, a green alga and a higher plant. Cytologia 50: 75–82.
• Nishimura, Y., Higashiyama, T., Suzuki, R., Misumi, O. and Kuroiwa, T. 1998. The biparental transmission of the mitochondrial genome in Chlamydomonas reinhardtii visualized in living cells. Eur. J. Cell Biol. 77: 124–133.
• Nozaki, H., Takano, H., Misumi, O., Terasawa, K., Matsuzaki, M., Maruyama, S., Nishida, K., Yagisawa, F., Yoshida, Y., Fujiwara, T., Takio, S., Tamura, K., Chung, S. J., Nakamura, S., Kuroiwa, H., Tanaka, K., Sato, N. and Kuroiwa, T. 2007. A 100%-complete sequence reveals unusually simple genomic features in the hot-spring red alga Cyanidioschyzon merolae 10D. BMC Biol. 5: 28.
• Ohta, N., Matsuzaki, M., Misumi, O., Miyagishima, S., Nozaki, H., Kohara, Y. and Kuroiwa, T. 2003. Complete sequence and analysis of the plastid genome of the unicellular red alga Cyanidioschyzon merolae. DNA Res. 10: 67–77.
• Ohta, N., Sato, N. and Kuroiwa, T. 1998. Structure and organization of the mitochondrial genome of the unicellular red alga Cyanidioschyzon merolae deduced from the complete nucleotide sequence. Nucleic Acids Res. 26: 5190–5198.
• Ohta, N., Suzuki, S., Kawano, S. and Kuroiwa, T. 1993. Direct evidence of mitochondrial nuclear division in the ultra-micro alga Cyanidioschyzon merolae. Cytologia 58: 471–476.
• Sakai, A., Takano, H. and Kuroiwa, T. 2004. Structure, composition, function, and evolution of the organelle-nuclei in higher plants. Int. Rev. Cytol. 23: 860–107.
• Sando, N., Miyakawa, I., Nishibayashi, S. and Kuroiwa, T. 1981. Arrangement of mitochondrial nucleoids in life cycle of Saccharomyces cerevisiae. J. Gen. Appl. Microbiol. 27: 511–516.
• Sasaki, N., Kuroiwa, H., Nishitani, C., Takano, H., Higashiyama, T., Kobayashi, T., Shirai, Y., Sakai, A., Kawano, S., Murakami-Murofushi, K. and Kuroiwa, T. 2003. Glom is a novel mitochondrial DNA packaging protein in Physarum polycephalum and causes intense chromatin condensation without suppressing DNA functions. Mol. Biol. Cell 14: 4758–4769.
• Sasaki, N., Sakai, A., Kawano, S., Kuroiwa, H. and Kuroiwa, T. 1998. DNA synthesis in isolated mitochondrial nucleoids from plasmodia of Physarum polycephalum. Protoplasma 203: 221–231.
• Satoh, M. and Kuroiwa, T. 1991. Organization of multiple nucleoids and DNA molecules in mitochondria of a human cell. Exp. Cell Res. 196: 137–140.
• Seckbach, J. 1995. Origin and structure of the cell: Biological aspects the first eukaryotic cells-acid hot-spring algae. Evolutionary paths from prokaryotes to unicellular red algae via Cyanidium caldarium (PreRhodophyta) succession. J. Biol. Phys. 20: 335–345.
• Suzuki, K., Ehara, T., Osafune, T., Kuroiwa, H., Kawano, S. and Kuroiwa, T. 1994. Behavior of mitochondria, chloroplasts and their nuclei during the mitotic cycle in the ultramicroalga Cyanidioschyzone merolae. Eur. J. Cell Biol. 63: 280–288.
• Terui, S., Suzuki, K., Takahashi, H., Itoh, R. and Kuroiwa, T. 1995. Synchronization of chloroplast division in the ultramicroalga Cyanidioschyzon merolae (Rhodophyta) by treatment with light and aphidicolin. J. Phycol. 31: 58–961.
• Toda, K., Takahashi, H., Itoh, R. and Kuroiwa, T. 1995. DNA contents of cell nuclei in two Cyanidiophyceae: Cyanidioschyzon merolae and Cyanidium caldarium forma A. Cytologia 60: 183–188.
• Whitaker R. J., Grogan, D. W. and Taylor, J. W. 2005. Recombination shapes the natural population structure of the hyperthermophilic archaeon Sulfolobus islandicus. Mol. Biol. Evol. 22: 2354–2361.
• Yagisawa, F., Fujiwara, T., Takemura, T., Kobayashi, K., Sumiya, N., Miyagishima, S. Y., Nakamura, S., Imoto, Y., Misumi, O., Tanaka, K., Kuroiwa, H. and Kuroiwa, T. 2020. ESCRT machinery mediates cytokinetic abscission in the unicellular red alga Cyanidioschyzon merolae. Front. Cell Dev. Biol. 8: A169.
• Yagisawa, F., Imoto, Y., Fujiwara, T. and Miyagishima, S. 2017. Single Membrane-Bound Organelles: Division and Inheritance. In: Kuroiwa, T., Miyagishima, S. Y., Matsunaga, S., Sato, N., Tanaka, K. and Misumi, O. (eds.). Cyanidioschyzon merolae. Springer Nature, Singapore. pp. 235–249.
• Yoshida, Y., Kuroiwa, H., Misumi, O., Nishida, K., Yagisawa, F., Fujiwara, T., Nanamiya, H., Kawamura, F. and Kuroiwa, T. 2006. Isolated chloroplast division machinery can actively constrict after stretching. Science 313: 1435–1438.
• Yoshida, Y., Kuroiwa, H., Misumi, O., Yoshida, M., Ohnuma, M., Fujiwara, T., Yagisawa, F., Hirooka, S., Imoto, Y., Matsushita, K., Kawano, S. and Kuroiwa, T. 2010. Chloroplasts divide by contraction of a bundle of nanofilaments consisting of polyglucan. Science 329: 949–953.
• Yoshida, Y., Kuroiwa, H., Shimada, T., Yoshida, M., Ohnuma, M., Fujiwara, T., Imoto, Y., Yagisawa, F., Nishida, K., Hirooka, S., Misumij, O., Mogi, Y., Akakabe, Y., Matsushita, K. and Kuroiwa, T. 2017. Glycosyltransferase MDR1 assembles a dividing ring for mitochondrial proliferation comprising polyglucan nanofilament. Proc. Natl. Acad. Sci. U.S.A. 114: 13284–13289.
• Zaremba-Niedzwiedzka, K., Caceres, E. F., Saw, J. H., Bäckström, D., Juzokaite, L., Vancaester, E., Seitz, K. W., Anantharaman, K., Starnawski, P., Kjeldsen, K. U., Stott, M. B., Nunoura, T., Banfield, J. F., Schramm, A., Baker, B. J., Anja Spang, A. J. and Ettema, T. J. G. 2017. Asgard archaea illuminate the origin of eukaryotic cellular complexity. Nature 541: 353–358.