Isolation of dividing chloroplasts with intact plastid-dividing rings from a synchronous culture of the unicellular red alga Cyanidioschyzon merolae

Miyagishima, Shin-ya; Itoh, Ryuuichi; Aita, Saburo; Kuroiwa, Haruko; Kuroiwa, Tsuneyoshi

doi:10.1007/s004250050645

Cited by 53 publications

(51 citation statements)

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“…To isolate dividing and nondividing chloroplasts, we harvested cells at the middle of the second dark and third light periods, respectively. In some experiments, to obtain highly synchronized M phase culture, we treated the cells with 5-fluorodeoxyuridine (Sigma) at a concentration of 10 g/mL at the onset of the second dark period (Miyagishima et al, 1999c). The nondividing chloroplasts also were isolated from concentrated culture ‫3ف(‬ ϫ 10 7 cells/mL), in which cells did not divide even under the light/dark cycles.…”

Section: Isolation Of Dividing and Nondividing Chloroplasts From Syncmentioning

confidence: 99%

“…The nondividing chloroplasts also were isolated from concentrated culture ‫3ف(‬ ϫ 10 7 cells/mL), in which cells did not divide even under the light/dark cycles. The chloroplasts were isolated from synchronized culture, as described previously (Miyagishima et al, 1999c). Isolated chloroplasts suspended in isolation medium (20 mM Tris, pH 7.6, 5 mM MgCl 2 , 5 mM KCl, 5 mM EGTA, and 300 mM sucrose) were kept on ice until use.…”

Section: Isolation Of Dividing and Nondividing Chloroplasts From Syncmentioning

confidence: 99%

“…For this purpose, we isolated dividing chloroplasts with PD rings (Miyagishima et al, 1999c) and observed the outer ring by negative staining with the aid of a detergent. We found that the main constituent of the outer PD ring is a bundle of novel filaments other than FtsZ that is 5 nm in diameter.…”

Section: Introductionmentioning

confidence: 99%

“…However, in this study, any helical structure similar to F-actin could not be detected around the outer PD ring. Isolated dividing chloroplasts did not stain with FITC-phalloidin (Miyagishima et al, 1999c). In red alga Cyanidium caldarium, the contractile ring was labeled by anti-actin antibody by using immunoelectron microscopy , but the PD ring was not (H. Takahashi, S. Miyagishima, T. Kuroiwa, unpublished data).…”

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confidence: 99%

“…Chloroplasts were treated with several detergents, and chloroplasts and outer PD rings were viewed by fluorescence microscopy. The outer PD ring was visualized by labeling surface proteins of chloroplasts with N -hydroxy-sulfo-succinimidyl (NHS)-biotin and detected by fluorescein isothiocyanate (FITC)-avidin, as described previously (Miyagishima et al, 1999c) (Figure 1D). …”

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Novel Filaments 5 nm in Diameter Constitute the Cytosolic Ring of the Plastid Division Apparatus

Miyagishima¹,

Takahara²,

Kuroiwa³

2001

The Plant Cell

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The plastid division apparatus (called the plastid-dividing ring) has been detected in several plant and algal species at the constricted region of plastids by transmission electron microscopy. The apparatus is composed of two or three rings: an outer ring in the cytosol, an inner ring in the stroma, and a middle ring in the intermembrane space. The components of these rings are not clear. FtsZ, which forms the bacterial cytokinetic ring, has been proposed as a component of both the inner and outer rings. Here, we present the ultrastructure of the outer ring at high resolution. To visualize the outer ring by negative staining, we isolated dividing chloroplasts from a synchronized culture of a red alga, Cyanidioschyzon merolae , and lysed them with nonionic detergent Nonidet P-40. Nonidet P-40 extracted primarily stroma, thylakoids, and the inner and middle rings, leaving the envelope and outer ring largely intact. Negative staining revealed that the outer ring consists of a bundle of 5-nm filaments in which globular proteins are spaced 4.8 nm apart. Immunoblotting using an FtsZ-specific antibody failed to show immunoreactivity in the fraction containing the filament. Moreover, the filament structure and properties are unlike those of known cytoskeletal filaments. The bundle of filaments forms a very rigid structure and does not disassemble in 2 M urea. We also identified a dividing phase-specific 56-kD protein of chloroplasts as a candidate component of the ring. Our results suggest that the main architecture of the outer ring did not descend from cyanobacteria during the course of endosymbiosis but was added by the host cell early in plant evolution. INTRODUCTIONEukaryotic cells contain organelles that are duplicated and inherited by daughter cells during cell division. Among these organelles, mitochondria and plastids are unique in that they contain DNA-protein complexes (nucleoids) and machinery sufficient for protein synthesis. It is generally believed that mitochondria and plastids arose from prokaryotic endosymbionts during eukaryotic evolution. The endosymbiotic theory states that the progenitor of mitochondria was an ␣ -proteobacterium and that the plastid ancestor was a cyanobacterium (Gray, 1992). Mitochondria and plastids multiply by the division of preexisting organelles, as do bacteria (Leech et al., 1981;Kuroiwa, 1982Kuroiwa, , 1991Possingham and Lawrence, 1983; Boffey and Lloyd, 1988). Of the characterized mitochondrial and plastid genomes, however, none has a complete set of genes sufficient for self-replication. Mitochondria that cannot synthesize proteins as a result of large genome deletions (petite mutant) (Attardi and Schatz, 1988) and plastids that lack ribosomes (Hashimoto and Possingham, 1989) still can proliferate. Therefore, it is believed that products from the host (nuclear) genomes perform mitochondrial and plastid division. Nuclear regulation of organelle division remains poorly understood, but in plastid division both the plastid-dividing ring (PD ring) (summarized in Kuroiwa et ...

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Section: Isolation Of Dividing and Nondividing Chloroplasts From Syncmentioning

confidence: 99%

Section: Isolation Of Dividing and Nondividing Chloroplasts From Syncmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Novel Filaments 5 nm in Diameter Constitute the Cytosolic Ring of the Plastid Division Apparatus

Miyagishima¹,

Takahara²,

Kuroiwa³

2001

The Plant Cell

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Cell Growth Control in an Algal Model

Bišová

Plant Cell Monographs

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In the Grip of Algal Genomics

Grossman

Transgenic Microalgae as Green Cell Factories

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Algae are dominant primary producers on the Earth and have a major impact on global productivity and biogeochemical cycling. There are still few algal genomes that have been completely characterized, and resources directed toward algal genomic sequencing are limited. However, it is also becoming evident that algae and prokaryotic picoplankton have a critical role in the fixation and sequestration of carbon, and so the interest in algal genomics is expanding. There are some algae for which full or near-full genome sequences have been secured; these genomes include those of the red alga Cyanidioschyzon merolae, the green algae or chlorophytes Chlamydomonas reinhardtii and Volvox carteri, the marine picoeukaryote Ostreococcus tauri (two different strains of O. tauri have been sequenced), the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum, and the haptophyte Emiliania huxleyi. There is also a full sequence for the vestigal 'red' algal genome of the nucleomorph of the Cyptomonad Guillardia theta. In addition, numerous genomes of photosynthetic microbes, including marine Synechococcus and Prochlorococcus species have been sequenced. There have also been projects developed to define algal transcriptomes as determined by cDNA analysis, full genome sequences of numerous plastids, and the genomes of a variety of viruses that infect marine and freshwater algae. The recent efforts focused on acquiring and analyzing algal genome sequences have generated an influx of exciting data to a field that is in its infancy. In this review I discuss potential criteria for determining which organisms should be targeted for genome projects, successful forays into algal genomic sequencing, and some of the inferences generated from the analysis of the sequence information.

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Isolation of dividing chloroplasts with intact plastid-dividing rings from a synchronous culture of the unicellular red alga Cyanidioschyzon merolae

Cited by 53 publications

References 22 publications

Novel Filaments 5 nm in Diameter Constitute the Cytosolic Ring of the Plastid Division Apparatus

Novel Filaments 5 nm in Diameter Constitute the Cytosolic Ring of the Plastid Division Apparatus

Cell Growth Control in an Algal Model

In the Grip of Algal Genomics

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