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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2008 Sep 22;105(39):15202–15207. doi: 10.1073/pnas.0802412105

Evolutionary linkage between eukaryotic cytokinesis and chloroplast division by dynamin proteins

Shin-ya Miyagishima *,†, Hidekazu Kuwayama ‡, Hideko Urushihara ‡, Hiromitsu Nakanishi *
PMCID: PMC2567515  PMID: 18809930

Abstract

Chloroplasts have evolved from a cyanobacterial endosymbiont and been retained for more than 1 billion years by coordinated chloroplast division in multiplying eukaryotic cells. Chloroplast division is performed by ring structures at the division site, encompassing both the inside and the outside of the two envelopes. A part of the division machinery is derived from the cyanobacterial cytokinetic activity based on the FtsZ protein. In contrast, other parts of the division machinery involve proteins specific to eukaryotes, including a member of the dynamin family. Each member of the dynamin family is involved in the division or fusion of a distinct eukaryotic membrane system. To gain insight into the kind of ancestral dynamin protein and eukaryotic membrane activity that evolved to regulate chloroplast division, we investigated the functions of the dynamin proteins that are most closely related to chloroplast division proteins. These proteins in the amoeba Dictyostelium discoideum and Arabidopsis thaliana localize at the sites of cell division, where they are involved in cytokinesis. Our results suggest that the dynamin for chloroplast division is derived from that involved in eukaryotic cytokinesis. Therefore, the chloroplast division machinery is a mixture of bacterial and eukaryotic cytokinesis components, with the latter a key factor in the synchronization of endosymbiotic cell division with host cell division, thus helping to establish the permanent endosymbiotic relationship.

It is widely believed that chloroplasts arose from a bacterial endosymbiont related to extant cyanobacteria (1, 2). Although most of their genes have either been lost or transferred to the host nuclear genome, chloroplasts retain several features similar to cyanobacteria. Chloroplasts contain nucleoids and ribosomes, and they are not synthesized de novo (1, 2). Chloroplasts multiply by division, as do cyanobacteria (3). However, the chloroplast genome does not contain sufficient information for carrying out division, indicating that the host eukaryotic cell genome regulates the division of chloroplasts (3).

Chloroplast division is performed by the constriction of a division apparatus (ring) encircling the division site around the two envelope membranes (3–6). The division apparatus includes a plastid-dividing ring of unknown composition, FtsZ, and one of the dynamin family of proteins (4–6). FtsZ and its associated factors are descended from the cyanobacterial endosymbiont, posttranslationally targeted into chloroplasts (4). In contrast, the dynamin family of GTPases is specific to eukaryotes, and the chloroplast division dynamin is recruited to the cytosolic side of the chloroplast division site (7–9). This suggests that the chloroplast division machinery is derived from both endosymbiotic (bacterial) and host (eukaryotic) cells.

The cyanobacteria-descended components of the chloroplast division machinery evolved from the cell division machinery of the cyanobacterial endosymbiont (4, 5). In contrast, there is little information about the origin of chloroplast division dynamin proteins. The dynamin family of GTPase proteins self-assemble into rings or spirals on the surface of eukaryotic membranes, where they play roles in membrane fission or fusion (10). There are divergencies in the dynamin family, and the function of each member has been assigned to a distinct eukaryotic membrane activity, such as transport vesicle budding, organelle division, cytokinesis, and pathogen resistance (10). In some cases, two or more functions have been assigned to the same protein (10). Among the dynamin family, chloroplast division proteins specifically localize at the chloroplast division site (7–9), and mutations specifically inhibit chloroplast division (8, 11), suggesting that the proteins function exclusively in chloroplast division in extant plants and algae. However, given that the dynamin family already existed in eukaryotes before the emergence of chloroplasts (4, 5, 10), the dynamin proteins involved in chloroplast division probably are derived from those involved in eukaryotic membrane systems other than that in the chloroplast. Understanding the eukaryotic membrane fission/fusion machinery, which has evolved into the division mechanism of organelles, should provide important insights into the question of how host cells have regulated the division of endosymbionts to establish a permanent endosymbiotic relationship. It has been suggested that synchronization of the host–endosymbiont cell cycle and cosegregation are critical steps (12, 13), but it is not known how the synchronization was established in ancestral algae.

In this study, we found that previously uncharacterized members of the dynamin family in plants and nonphotosynthetic protists share a common ancestor with the plant-specific chloroplast division dynamin proteins. Our results show that these proteins of amoeba and plants are involved in eukaryotic cytokinesis. These results suggest that the dynamin used in chloroplast division is derived from that involved in eukaryotic cytokinesis. Application of cytokinetic dynamin to endosymbiont cell division may have enabled the synchronization of host–endosymbiont cell division such that each daughter cell can inherit an endosymbiont after cytokinesis.

Results

Phylogenetic Relationships in the Dynamin Family.

To address the questions of the type of dynamin family member that evolved into the chloroplast division protein and the membrane activity in which the ancestor of the chloroplast division dynamin was involved, we conducted phylogenetic analyses of the dynamin family. In these phylogenetic analyses, we included both previously characterized and uncharacterized members of the dynamin family of Plantae, Opisthokonta (fungi and animals), Amoebozoa, Heterolobosea, Chromista (a stramenopile), and Alveolata (ciliates). Because the dynamin family has a diverse range of functions (10), it is expected that the evolutionary rates vary with the specific functions. To avoid the generation of an incorrect tree, which is commonly a problem with rapidly evolving sequences (long-branch attraction artifacts), we constructed phylogenetic trees by maximum-likelihood methods using amino acid sequences [ProtML (14) and PhyML (15)].

The phylogenetic analyses showed a distant relationship between chloroplast division DRP5B proteins (grouped in green in Fig. 1; originally named CmDnm2 in the red alga Cyanidioschyzon merolae and named ARC5 in Arabidopsis thaliana; later renamed DRP5B, ref. 16) and other characterized members of the dynamin family (Fig. 1). However, the phylogenetic tree revealed that the chloroplast division proteins are most closely related to certain uncharacterized proteins (grouped in purple in Fig. 1) of plants and protists that do not have chloroplasts. These uncharacterized members are three of the five Dictyostelium discoideum (Amoebozoa) dynamin proteins, named DlpA, DlpB and DlpC, and plant and algal DRP5A proteins, in agreement with previous partial annotations (10, 16). In addition, a putative protein, Dnm2 of Naegleria gruberi (Heterolobosea), was included in the uncharacterized group. Using BLAST searches of databases, we also found two putative proteins similar to DRP5A in the amoeba Entamoeba histolytica (Amoebozoa, GenBank accession numbers XP_653348 and XP_651307, omitted from the phylogenetic analyses because these made very long branches) and the expressed sequence tags from Histiona aroids (Jakobidae, GenBank accession numbers EC851519 and EC850393) encoding a protein similar to DRP5A.

Fig. 1.

Fig. 1.

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Phylogenetic relationships in the dynamin family of proteins. The tree shown is the maximum-likelihood tree constructed by the PhyML program based on alignment of 567 amino acid residues of 78 dynamin family proteins. The numbers at the nodes are local bootstrap values calculated using PhyML and ProtML analyses. Bootstrap values >80% are shown, and dashes indicate <80% support. Branch lengths are proportional to the number of amino acid substitutions, which are indicated by the scale bar below the tree. The tree includes the dynamin family of proteins of Plantae Arabidopsis thaliana (At), Oryza sativa (Os or locus ID start from Os), Chlamydomonas reinhardtii (Cr), Ostreococcus lucimarinus (Ot), Cyanidioschyzon merolae (Cm), Stramenopile Thalassiosira pseudonana (Tp), Amoebozoa Dictyostelium discoideum (Dd), Heterolobosea Naegleria gruberi (Ng) Alveolata Tetrahymena thermophila (Tt), Paramecium tetraurelia (Pt), Opisthokonta Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), Homo sapiens (Hs), Drosophila melanogaster (Dm), and Caenorhabditis elegans (Ce).

The monophyly of plant and algal DRP5A (Fig. 1, branch d), that of plant and algal chloroplast division DRP5B (Fig. 1, branch e), and that of amoeba DlpA and DlpB (Fig. 1, branch c) were strongly supported by the bootstrap values of 100/100 (PhyML/ProtML). The monophyly of plant and algal DRP5A, amoeba DlpA, and the heterolobosean Dnm2 was also supported by the bootstrap values of 82/92 (Fig. 1, branch b). Finally, our phylogenetic analyses suggest the monophyly of all of the above-mentioned proteins (Fig. 1, branch a) by the bootstrap values of 100/100. These results suggest that the chloroplast division proteins DRP5A and the uncharacterized proteins share a common ancestor.

The uncharacterized members of the dynamin family (grouped in purple in Fig. 1) are widespread in eukaryotes, including plants and algae, and three other eukaryotic groups which do not have chloroplasts (Amoebozoa, Heterolobosea, and Jakobidae). Previous phylogenetic studies suggested that Amoebozoa, Heterolobosea, and Jakobidae had diverged from an ancestor of plant cells before the acquisition of the chloroplast (2, 17). Taken together, these results suggest that chloroplast division dynamin proteins, which are specific to plant and algae, derived from an ancestor of the uncharacterized members that is commonly shared by photosynthetic and nonphotosynthetic eukaryotes.

DlpA, DlpB, and DlpC Are Involved in Cytokinesis in the Amoeba D. discoideum.

To address the functions of the dynamin proteins from which the chloroplast division proteins were derived, we examined the functions of DlpA, DlpB, and DlpC in D. discoideum. When the genes were disrupted by homologous recombination, ΔdlpA, ΔdlpB, and ΔdlpC mutants produced cells larger than those of the wild type, and a large population of the mutant cells contained more than two nuclei (in contrast to wild-type cells containing one or two duplicated nuclei) (Fig. 2A), similar to the known cytokinetic mutants of D. discoideum (18). These results indicate that nuclear division occurs normally but cytokinesis is defective in the mutant cells. Since single-gene mutations have an affect on cytokinesis, it is suggested that the functions of DlpA, DlpB, and DlpC are not redundant and are distinct in the activity of cytokinesis.

Fig. 2.

Fig. 2.

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DlpA, DlpB, and DlpC are involved in cytokinesis in D. discoideum. (A) The ΔdlpA, ΔdlpB, and ΔdlpC mutant amoeba cells (observed using phase-contrast microscopy) are multinucleated (nuclei are shown with blue fluorescence by DAPI staining) and larger than the wild-type (WT) cells. (B) Time course of dlpA, dlpB, and dlpC mRNA and DlpA protein levels during spore germination and cell culture mRNA were detected by semiquantitative RT-PCR cycB (encoding G2/M-specific cyclin B). The expression level indicates the frequency of cells in M phase. TFIIS (encoding transcription elongation factor IIS) was used as an internal control. The DlpA protein was detected by anti-DlpA antibodies, which specifically detect DlpA, as shown at the Left. The lowermost bands of Ponceau S staining show the same amount of total protein was loaded in each lane. (C) Immunofluorescence images showing that DlpA localizes at the cleavage furrow during cytokinesis. Green fluorescence corresponding to anti-DlpA antibodies and phase-contrast images of amoeba are overlaid. Orange fluorescence corresponding to the microtubules stained by the anti-α-tubulin antibody, and blue fluorescence showing nuclei are indicators of stages of the cell cycle: I1, interphase; M, metaphase indicated by the short spindle; A, anaphase indicated by the elongated spindle; T1, T2, and T3, telophase indicated by the cleavage furrow; and I2, interphase just after cytokinesis. (Scale bars: A, 20 μm; C, 10 μm.)

To examine the relationship between the expression of dlpA, dlpB, and dlpC and cell division, we germinated spores of D. discoideum and compared the mRNA levels of the dlpA, dlpB, and dlpC and DlpA proteins during cell cycle progression after germination (Fig. 2B). After synchronous germination of the wild-type spores was induced by heat shock treatment (19), spores germinated within 8 h and amoebas in the G1 phase (19) appeared. Cells started to divide after 24 h and reached a stationary phase (G2 phase) (19) between 72 and 96 h. Like the case of the G2/M cyclin gene (cycB), dlpA, dlpB, and dlpC gene expression begins between 12 and 24 h. Immunoblot analysis using anti-DlpA antibodies showed that the DlpA protein appeared at the onset of log-phase (24 h), reached a maximum level around mid–log-phase, and then disappeared during the stationary phase (Fig. 2B). These results suggest that the DlpA protein expresses specifically during the M phase and that the protein level is regulated by transcription and protein degradation according to the phase of the cell cycle.

To examine whether DlpA is directly involved in cytokinesis, we examined the localization of DlpA throughout the cell cycle. Immunofluorescence microscopy using anti-DlpA antibodies revealed that DlpA localizes at the cleavage furrow during cytokinesis (Fig. 2C). DlpA localization was detected at the cleavage furrow in cells during telophase and at sites where cells had divided (Fig. 2C). The above results indicate that DlpA, and probably DlpB and DlpC, are directly involved in cytokinesis in D. discoideum.

DRP5A Is Involved in Cytokinesis in A. thaliana.

DRP5A proteins in plants and algae were predicted to be involved in chloroplast division based on the similarity between DRP5A and chloroplast division DRP5B proteins (16). However, the above results showing the involvement of DlpA in cytokinesis in D. discoideum and the monophyly of DlpA, plant and algal DRP5A, and chloroplast division DRP5B proteins (Fig. 1, branch a) raised the possibility that DRP5A proteins are involved in cytokinesis rather than chloroplast division.

To examine the function of plant DRP5A proteins, we expressed a DRP5A-GFP fusion protein using the DRP5A promoter in A. thaliana. DRP5A-GFP signals were detected in meristematic tissues (root tips are shown in Fig. 3 A and B) and meristemoid cells in the leaf epidermis (Fig. 3C), but no signal was detected in other somatic cells or chloroplast division sites. These observations suggest that DRP5A expression is limited to dividing cells and that DRP5A is involved in activities other than chloroplast division. In the root tip, the fluorescent signal was limited to a certain number of cells (Fig. 3 A and B; the two cells at the lower left are fluorescing, but the others are not in Fig. 3B), with this mosaic pattern being similar to the expression patterns of cell cycle–regulated proteins, such as cyclin B (20). Data from the Genevestigator site (http://www.genevestigator.ethz.ch) show M phase–specific accumulation of the DRP5A mRNA detected by cell cycle synchronization in an A. thaliana cultured cell line (21). When DRP5A-GFP–expressing plants were treated with colchicine to arrest the cell cycle at the M phase, most of the root tip cells displayed fluorescence (Fig. 3D). In contrast, aphidicolin treatment to arrest the cell cycle at the S phase resulted in little fluorescent signal (Fig. 3D). These results suggest that DRP5A protein expression is also specific to the M phase, and the protein level is controlled, at least in part, by the transcription level and the degradation of the proteins after the M phase.

Fig. 3.

Fig. 3.

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DRP5A is involved in cytokinesis in A. thaliana. (A) DRP5A-GFP expression by the DRP5A promoter (Right) showing the presence of DRP5A in some cells of a root tip (Left, differential interference contrast image). (B) A magnified image of the root tip showing patchy localization and bar-shaped localization of DRP5A-GFP in two distinct cells and no signal in other cells. (C) DRP5A-GFP exhibits patchy signals in the meristemoid of leaf epidermis but exhibits no signal in other epidermal cells, including guard cells. (D) Colchicine-mediated (+col) arrest of root cells in the M phase reveals DRP5A-GFP expression in all cells, but S-phase arrest by aphidicolin (+aph) abolishes DRP5A-GFP expression. (E) Immunofluorescence images showing DRP5A localization at cell plates during cytokinesis. DRP5A localization in root tip cells (Left) and a series of images showing the transition of DRP5A localization during the cell cycle (Right). Green fluorescence indicates DlpA localization by the anti-DRP5A antibodies. Orange fluorescence indicates microtubules by the anti-α-tubulin antibody, blue fluorescence by DAPI staining indicates nuclei, and differential interference contrast images are indicators of the stages of cell cycle. I, interphase; P, prophase indicated by the preprophase band of microtubules, M, metaphase indicated by the spindle; T1, early telophase indicated by the expanding phragmoplast; T2, midtelophase indicated by the expanded phragmoplast; T3, late telophase. (F) Positions of T-DNA insertion of drp5A-1 (No-0 background) and drp5A-2 (Col-0 background). Exons are depicted as rectangles. (G–I) Phenotypes of drp5A-2 seedlings compared with those of the wild type (Col-0) at 16°C. The same results were obtained in drp5A-1 seedlings compared with No-0 wild-type plants. (G) Ten-day-old wild-type (WT) and drp5A-2 seedlings. (H and I) Shown are disarrangement of root tip cells in drp5A-2 (H) and incomplete (Lower Left, arrow) or disordered (Lower Right, arrows) formation of cell plates in drp5A-2 (I). (Scale bars: A, C, D, and H, 20 μm; B, E, and I, 10 μm.)

In the root tip, speckles of fluorescence were observed in some cells, but a bar-shaped localization pattern was observed in other cells (Fig. 3B), suggesting that the localization of DRP5A changes during the cell cycle. To verify whether DRP5A-GFP reflects the true localization of endogenous DRP5A, and to examine the transition of DRP5A localization during the cell cycle, we performed immunofluorescence microscopy using anti-DRP5A antibodies. In keeping with the DRP5A-GFP fusion protein results, the antibody results showed speckles and bar-shaped structures (Fig. 3E). Simultaneous labeling of tubulin and DNA showed the transition of DRP5A localization during the cell cycle (Fig. 3E). Interphase cells showed no specific signal, whereas in the prophase, speckles of DRP5A were detected around the nucleus. The speckles were dispersed in the cytosol of cells in the metaphase or early telophase. In late-telophase cells, most of the speckles were detected at the cell plate, whereas at the end of cell division, DRP5A was detected uniformly at the cell plate. These results suggest that DRP5A plays a role in cytokinesis.

To confirm that DRP5A is required for cytokinesis rather than chloroplast division, we examined two independent insertional mutant lines of DRP5A (Fig. 3F). Although the mutant lines grew normally when seeds were germinated under conventional conditions (21°C), mutant seedlings grew more slowly than those of the wild type at a lower temperature (16°C) (Fig. 3G). In both conditions, the chloroplast size and number per cell in the dlp5A mutants were normal, whereas the chloroplast division dlp5B mutant cells contained chloroplasts that were fewer in number and larger than in the wild type (8, 9, 11). Microscopic examination of the mutant root tips revealed perturbation of the cell array (Fig. 3H) and formation of incomplete or twisted cell plates in mutant cells (Fig. 3I), similar to other A. thaliana mutants of cytokinetic proteins (22). The same phenotypes were observed in two independent drp5A alleles, suggesting that the observed effects are caused by mutations in the gene. These results suggest that loss of DRP5A affected cell plate formation and that, rather than chloroplast division, DRP5A is involved in cytokinesis, at least under lower temperature conditions, which often occur in the wild.

The lack of detectable phenotypes in the drp5A mutants at normal temperatures may be caused by redundant or overlapping functions provided by other proteins. The genome of A. thaliana has only a single copy of the DRP5A gene, and the most closely related protein of DRP5A is DRP5B. We reexamined GFP-DRP5B localization in A. thaliana (8, 9), and the localization was specific to the chloroplast division site, as previously observed (8, 9), whereas no localization was observed around the cell plate. Also, we reexamined the phenotypes of the drp5B mutants (8, 9, 11), but the pattern of cell division was normal. These results suggest that the function of DRP5B is specific to chloroplast division.

Arabidopsis thaliana contains several members of the DRP1 and DRP2 cytokinetic dynamin proteins (16, 23), although they are evolutionarily distantly related to DRP5A (Fig. 1). To examine the potential existence of functional redundancy, we overexpressed DRP5A-GFP or DRP5A (K75A), which corresponds to K44A dominant-negative mutant of human dynamin 1. However, the overexpressers grew normally, and the cell division patterns were normal (data not shown). Although at present it is unclear why the cytokinetic defects of the drp5A mutant appear only at a lower temperature, the exclusive expression of DRP5A in meristematic cells during the M phase along with the cell plate localization of DRP5A suggest a function in cell division.

Discussion

Evolutionary Linkage Between Cytokinesis and Chloroplast Division.

Every member of the dynamin family is involved in fission or fusion of distinct eukaryotic membrane systems, including the division of endosymbiotic organelles, mitochondria, and chloroplasts (4, 5, 10). The question addressed by this study is the kind of dynamin that evolved into the dynamin proteins specific to chloroplast division, and the eukaryotic membrane activity that evolved into the chloroplast division machinery after the endosymbiosis of a cyanobacterium.

We have shown that chloroplast division DRP5B proteins share a common ancestor with previously uncharacterized members of the dynamin family, the function of which has been assigned by our analyses to cytokinesis both in the plant A. thaliana and the amoeba D. discoideum. The phylogenetic studies revealed a monophyly of the dynamin proteins involved in the chloroplast division and eukaryotic cytokinesis, and the existence of the latter in plants and algae, in addition to protists, which do not have chloroplasts. These results suggest that (i) a common ancestor of the chloroplast division and the cytokinetic dynamin proteins was involved in cytokinesis in ancestral nonphotosynthetic eukaryotes, and (ii) the chloroplast division DRP5B proteins were derived from the cytokinetic dynamin proteins.

Some members of the dynamin family possess multiple functions. For example, dynamin proteins in animals are involved in both endocytosis and cytokinesis (10, 24). The mitochondrial division dynamin proteins in certain organisms are also involved in peroxisomal division (10). Although cytokinetic DRP5A and chloroplast division DRP5B share a common ancestor, their completely distinct subcellular localization and nonoverlapping mutant phenotypes suggest that the functions of DRP5A and DRP5B are completely distinct in the extant plants, at least in A. thaliana.

It has been suggested that the synchronization of host–endosymbiont cell division is a critical step in the permanent fusion of the two partners (12, 13). There are eukaryotic species that contain transient photosynthetic endosymbionts called kleptoplasts in the cells. For example, Hatena arenicola has a transient green algal endosymbiont, and this photosynthetic endosymbiont is inherited by only one daughter cell during cell division (12). Hatena arenicola (Katablepharidophyta) bears a close evolutionary relationship with cryptophytes, in which permanent chloroplasts are established (25). A similar situation has been observed in Dinoflagellata (26) and Cercozoa (27), in which certain species have established the division synchronization, whereas others still have a transient endosymbiotic relationship with photosynthetic eukaryotes or cyanobacteria. The above observations suggest that division synchronization was necessary for the establishment of permanent chloroplasts (13). However, how this synchronization was established has remained largely unknown. Our results suggest that the host cells have made use of the M phase–specific cytokinetic dynamin proteins for the division of the endosymbiont. As a result, the division of the endosymbiont is limited to the M phase, so that each daughter cell inherits a daughter endosymbiont.

Notably, DRP5B chloroplast division dynamin is expressed specifically during the M phase in the primitive red alga Cyanidioschyzon merolae, which contains only a single chloroplast per cell, and therefore chloroplast division and cell cycle are synchronized in this alga (7). This is consistent with M phase–specific expression of DRP5A and Dlp proteins for cytokinesis (Figs. 2 and 3). However, the expression of the DRP5B chloroplast division protein in A. thaliana is apparently constant during the cell cycle, based on the experimental results of cell cycle synchronization in a cultured cell line (ref. 21; http://www.genevestigator.ethz.ch). In land plants, each cell contains more than one chloroplast, the division of which is not synchronous (28). Chloroplasts divide also in postmeristematic cells, which expand without cell division (28). These results suggest that cells with multiple chloroplasts have evolved more complex systems to regulate the expression of the chloroplast division dynamin proteins.

Evolution of the Dynamin Family.

To date, certain members of the dynamin family have been shown to be involved in cytokinesis (23, 24, 29, 30). In the phylogenetic tree, these proteins (members separately grouped in yellow and gray, DymA of D. discoideum in the mitochondrial division clade in Fig. 1), have a relatively close relationship (Fig. 1). The involvement of the amoeba DlpA (probably DlpB and possibly DlpC also) and plant DRP5A (grouped in purple in Fig. 1) in cytokinesis extends the cytokinetic function of the dynamin family throughout the phylogenetic tree, whereas members involved in other activities are grouped by their respective functions (Fig. 1). These results suggest that the original evolutionary function of dynamin proteins may have been involved in the cytokinesis of eukaryotic cells.

Plants and amoebae have several dynamin family members for cytokinesis (Fig. 1). Although the functions of some of these members may be partially redundant, single mutants of dlpA, dlpB, dlpC, and dlp5A display defects in cytokinesis, suggesting that the function of each of these proteins is not completely redundant with those of the others. Moreover, at least in the amoeba D. discoideum (DymA and the others grouped in purple) and plants and algae (grouped in gray and purple), there are evolutionarily distant cytokinetic dynamin proteins (Fig. 1). These facts suggest that at least two major groups of cytokinetic dynamins have already evolved to play different roles in the cytokinesis of ancestral eukaryotes.

Like chloroplasts, mitochondria evolved from an α-proteobacterial endosymbiont, and members of the dynamin family of proteins are involved in mitochondrial division (refs. 4, 5, and 10; and grouped in orange in Fig. 1). In addition, several lineages of eukaryotes still use the FtsZ descended from the α-proteobacterial endosymbiont to regulate mitochondrial division (31). The mitochondrion-dividing ring, which has a structure similar to the plastid-dividing (PD) ring, has been observed in some eukaryotic species (3, 5). These results suggest that the host cell used a strategy to regulate the division of the cyanobacterial endosymbiont similar to the one it used for the α-proteobacterial endosymbiont. Disruption of the dynamin gene dymA in D. discoideum blocks both cytokinesis and mitochondrial division (30). Similar results were also reported in a dynamin mutant of Trypanosoma brucei (32), although it is not known whether these dynamin proteins localize at the cleavage furrow. However, animal dynamin proteins (refs. 24 and 29; and grouped in yellow in Fig. 1) and plant-specific cytokinetic dynamin proteins (refs. 23 and 29; and grouped in gray in Fig. 1) were shown to localize at the site of cytokinesis, and these groups are closely related to the mitochondrial division group. These results raise the possibility that mitochondrial division dynamin proteins have evolved from the cytokinetic machinery of the host cell, as have chloroplast division proteins. DymA of D. discoideum (30) may represent an intermediate stage of evolution from the cytokinetic dynamin to that in endosymbiotic organelles. Investigation into the evolution of mitochondrial division dynamin proteins may elaborate the story of the chloroplast division dynamin protein to include another endosymbiotic organelle, mitochondrion.

Materials and Methods

Phylogenetic Analyses.

The maximum-likelihood tree was constructed with the PhyML program (15) using an alignment of amino acid sequences of 78 dynamin-like proteins [supporting information (SI) Table S1]. The sequences were collected by BLAST searches in the databases of respective species, National Center for Biotechnology Information, the US Department of Energy Joint Genome Institute (http://genome.jgi-psf.org/) using DRP5B and DRP3 of the red alga Cyanidioschyzon merolae as queries. The region of alignment contained the GTPase domain, middle domain, and GED domain (10) of the sequences. The local bootstrap probabilities were calculated using the PhyML (15) and ProtML (14) programs.

Cell Cultures and Plant Materials.

Wild-type D. discoideum (AX2) and the mutants derived from AX2 were cultured as previously described (33, 34). The dlpA, dlpB, and dlpC genes were disrupted by insertion of the blasticidin S-resistant gene via homologous recombination (33). Spores were germinated by heat shock as described previously (19), and DNase-treated total RNA was used for RT-PCR analyses. Wild-type A. thaliana, its transgenic lines, and homozygous drp5A T-DNA insertional mutants were grown as previously described (9), except where indicated. All plants were of Col-0 background, except for the phenotypic comparison between drp5A-1 and wild-type (Nos-0 background). The homozygous T-DNA insertion lines drp5A-1 (SALK_065118) and drp5A-2 (RATM12-3406-1_H) were provided by the Arabidopsis Biological Resource Center (ABRC) (35) and the RIKEN BioResource Center (36), respectively.

Immunoblotting and Immunofluorescence.

The 6× His-tag fusion of partial polypeptides of DlpA (511 residues) and DRP5A (445 residues) expressed in Escherichia coli was used to generate polyclonal antibodies in rabbits. Immunoblotting was performed using total proteins from amoeba cells, and immunofluorescence microscopy was performed on log-phase amoeba cells and root tips. The full experimental procedures and associated references are available in the SI Materials and Methods.

Supplementary Material

Supporting Information
supp_105_39_15202__index.html (628B, html)

Acknowledgments.

We thank C. Saito, T. Mori, and T. Kuroiwa for their help in microscopic analyses and for useful discussions, and we thank Y. Ono for technical support. We also thank the RIKEN BioResource Center and the Arabidopsis Biological Resource Center for providing seeds of drp5A-1 and drp5A-2. This work was supported by the Sumitomo Foundation (S.-y.M.)

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0802412105/DCSupplemental.

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