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Triple Immunofluorescent Labeling of FtsZ, Dynamin, and EF-Tu Reveals a Loose Association Between the Inner and Outer Membrane Mitochondrial Division Machinery in the Red Alga Cyanidioschyzon merolae

Keiji Nishida, Osami Misumi, Fumi Yagisawa, Haruko Kuroiwa, Toshiyuki Nagata and Tsuneyoshi Kuroiwa

Department of Life Science, College of Science, Rikkyo University (IKN,OM,FY,HK,TK), and Department of Biological Sciences, Graduate School of Science, University of Tokyo (KN,FY,TN), Tokyo, Japan

Correspondence to: Keiji Nishida, Dept. of Life Science, College of Science, Rikkyo University, Tokyo 171-8501, Toshima-ku, Japan. E-mail: keiji{at}platz.jp


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In the mitochondria of primitive eukaryotes, FtsZ and dynamin are part of the machinery involved in division of the inner and outer membranes, respectively. These genes also commonly function in the same manner during chloroplast division. In this study, a relationship between the localization of the inner and outer division machinery was directly shown for the first time. Triple immunofluorescent labeling was performed in the red alga Cyanidioschyzon merolae by a device using narrow bandpass filter sets and bright photostable dyes. FtsZ (CmFtsZ1) and dynamin (CmDnm1) localizations were examined simultaneously throughout the mitochondrial division cycle with an alternative mitochondrial marker protein, the mitochondrial translation elongation factor EF-Tu, whose localization was also shown to be identical to the mitochondrial matrix. FtsZ and dynamin did not necessarily co-localize when both were recruited to the mitochondrial constriction site, indicating that inner and outer dividing machineries are not in tight association during the late stage of division. (J Histochem Cytochem 52:843–849, 2004)

Key Words: FtsZ • dynamin • elongation factor • Tu • mitochondrial division • Cyanidioschyzon merolae • Alexa • triple immunofluorescence


    Introduction
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MITOCHONDRIAL DIVISION MACHINERY is now implicated in many processes, such as apoptosis and eukaryotic evolution as well as mitochondrial proliferation and inheritance. The machinery has been shown to have parts in common with that of the chloroplast (reviewed by Bossy-Wetzel et al. 2003Go; Miyagishima et al. 2003Go; Osteryoung and Nunnari 2003Go). Most mitochondria are so plastic, movable, and capable of dividing and fusing dynamically (reviewed by Yaffe 1999Go) that it is difficult to identify each of the phenomena independently. In this respect, the primitive red alga Cyanidioschyzon merolae is an ideal model for studying mitochondrial division because the cell contains only one mitochondrion of the simplest morphology, whose division can be easily synchronized by light and dark cycles (Suzuki et al. 1994Go), and the division apparatus, the mitochondrion-dividing (MD) ring, is visible with electron microscopy (reviewed by Kuroiwa et al. 1998Go). In addition, the genome of C. merolae has recently been sequenced, showing that C. merolae has a very simple gene composition and morphology for a eukaryote (Matsuzaki et al. 2004Go). Furthermore, both key proteins for mitochondrial division, FtsZ and dynamin, have been identified only in C. merolae, and neither protein is identical to the MD ring (Takahara et al. 2000Go; Nishida et al. 2003Go).

FtsZ is a self-assembling GTPase originally involved in bacterial cell division (reviewed by Margolin 2003Go), which was imported via endosymbiosis for use in the chloroplast (Osteryoung and Vierling 1995Go), and some primitive mitochondria (Beech et al. 2000Go; Takahara et al. 2000Go). FtsZs are required for normal division of the chloroplast (Osteryoung et al. 1998Go) or mitochondrion (Gilson et al. 2003Go) and form rings around the future division sites of chloroplasts (Mori et al. 2001Go; Vitha et al. 2001Go) and mitochondria (Beech et al. 2000Go; Nishida et al. 2003Go) as found in bacterial cells.

Dynamin, on the other hand, is a eukaryote-specific GTPase originally implicated in endocytotic vesicle formation. The dynamin family has now been found to be very diverse in function (reviewed by van der Bliek 1999Go). The involvement of dynamin in mitochondrial fission was first characterized in budding yeast as the protein Dnm1p (Otsuga et al. 1998Go; Bleazard et al. 1999Go) and was later found to be widespread in eukaryotes: humans (Smirnova et al. 1998Go), nematodes (Labrousse et al. 1999Go), higher plants (Arimura and Tsutsumi 2002Go), red algae (Nishida et al. 2003Go), and parasites (Morgan et al. 2003Go). These two dynamin homologues found in the C. merolae genome (Matsuzaki et al. 2004Go) have been characterized as dynamins involved in mitochondrial division (Nishida et al. 2003Go) and chloroplast division (Miyagishima et al. 2003Go). Arabidopsis thaliana also uses a dynamin for chloroplast division (Gao et al. 2003Go). In Trypanosoma brucei, only one dynamin, implicated in mitochondrial division but not involved in endocytosis, was found (Morgan et al. 2003Go). These findings further support the idea that dynamin in eukaryotes was originally involved in organelle division (Nishida et al. 2003Go). Dynamins involved in mitochondrial division are associated with the cytosolic surface of the outer membrane (Otsuga et al. 1998Go) and are involved in the fission of the outer but not the inner membrane (Labrousse et al. 1999Go). However, no counterpart on the inside of the division site has been found other than the mitochondrial FtsZ, which is not found in the genomes of most higher eukaryotes. Mitochondrial FtsZs have been identified in C. merolae (Takahara et al. 2000Go), Mallomonas splendens (Beech et al. 2000Go), and Dictyostelium discoideum (Gilson et al. 2003Go).

Therefore, C. merolae is the only organism whose mitochondrial machinery at both the inside and outside locations of the division site has been identified to date. In a previous study, we identified the localization of either FtsZ (CmFtsZ1) or dynamin (CmDnm1) independently by visualizing the mitochondria with Mitotracker CMX-Ros. In this study we characterized an alternative mitochondrial marker, EF-Tu, and developed a triple immunolabeling technique in the alga to reassess the localization of FtsZ and dynamin within the inner and outer division machineries.


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Antibodies and Immunoblotting Analysis
Mouse anti-CmFtsZ (Takahara et al. 2000Go) and rabbit anti-CmDnm1 (Nishida et al. 2003Go) antisera have been characterized previously. Rat anti-CmEF-Tu(mt) antiserum was generated as follows. An N-terminal truncated DNA fragment coding for residues 45–453 of CmEF-Tu(mt) (GenBank accession number AY573546; Matsuzaki et al. 2004Go) was amplified by PCR using the primers 5'-TAGGATCCACGACGTTGCAGTACCCG-3' and 5'-GAATTCAAGCTTCGATCTTTCGTAACC-3' and ligated into the pQE80 expression vector at the BamHI and HindIII sites. The ligated vector was transformed into E. coli XL1Blue MRF' and the expression of the recombinant protein containing a six-histidine tag at the N terminus was induced with 1 mM IPTG during mid-log phase of the culture. The cells were collected by centrifugation and denatured for 30 min in denaturing buffer containing 8 M urea, 10 mM phosphate buffer, pH 7.4, 200 mM NaCl, and 10% glycerol. The six-histidine-tagged protein was purified using a His-trap kit following the manufacturers' instructions (Amersham Biosciences; Piscataway, NJ) from the supernatant of the lysate. The purity of the protein was confirmed by SDS-PAGE electrophoresis and antibodies to the protein were generated by immunizing two rats (QIAGEN K.K.; Tokyo, Japan). Immunoblotting with anti-CmFtsZ1 and anti-CmDnm1 was performed as described (Nishida et al. 2003Go). For anti-CmEF-Tu(mt), briefly, 25 µg total protein of C. merolae was separated by 12.5% SDS-PAGE and blotted to polyvinylidene difluoride membrane. Immunoblotting was performed using the primary antiserum at a dilution of 1:1000 and the alkaline phosphatase-conjugated goat anti-rat, -rabbit, or -mouse IgG secondary antibody (KPL; Gaithersburg, MD) at a dilution of 1:1000. Signals were detected using an AP-conjugate substrate kit (Bio-Rad; Hercules, CA).

Cell Preparation
C. merolae cells were grown and synchronized as previously described (Suzuki et al. 1994Go). Cell fixation was performed as previously described (Nishida et al. 2003Go) with the following modifications. Cells were centrifuged for 5 min at 800 x g, then pre-fixed in 10 mM phosphate buffer (pH 7.5), 30 mM NaCl, 0.5 mM KCl, and 2% (w/v) paraformaldehyde for 3 min. Cells were then centrifuged for 3 min at 2000 x g for each buffer transfer and wash. Cells were gently suspended in the same pre-fixation buffer in a volume approximately equal to that of the cell pellet and then immediately fixed by addition of at least 20 volumes of chilled 85% (v/v) methanol, 15% (v/v) DMSO, 2% (w/v) paraformaldehyde, 1.5 mM NaOH solution for 5 min at –20C. The cells were then washed with methanol until completely dehydrated and rehydrated with PBS containing 137 mM NaCl, 2.7 mM KCl, 1 mM Na2HPO4, 1.4 mM K2HPO4. Cells were stored in PBS containing 50% (v/v) glycerol at –20C until use. If necessary, before fixation cells in growth medium were stained with Mitotracker CMX-Ros at a final concentration of 500 nM for 30 min to 1 hr until stained.

Indirect Immunofluorescence
Fixed cells were washed with PBS, blocked in 5% BSA in PBS at room temperature for 15 min, washed with 0.1% BSA in PBS, and then incubated with diluted primary antibodies in 0.1% BSA in PBS for 1 hr at 30C. After the primary antibody reaction, cells were washed twice with 0.1% BSA in PBS, then incubated with secondary antibodies diluted in a solution of 0.1% BSA in PBS for 30 min at 30C and then washed twice with 0.1% BSA in PBS. Primary and secondary antibodies were used at the following concentrations: 1:50 for rabbit anti-CmDnm1 antiserum, 1:100 for Alexa-350 goat anti-rabbit antibody, 1:100 for rat anti-CmEF-Tu(mt) antiserum, 1:200 for Alexa-555 goat anti-rat antibody or 1:100 for FITC goat anti-rat antibody, 1:20 for mouse anti-CmFtsZ1 antiserum, 1:1000 for Alexa-488 goat anti-mouse antibody.

Fluorescence Microscopy
Cells were observed using a fluorescence microscope, BX51 (Olympus), with a combination of narrow bandpass filter sets: BP360-370 BA420-460 (U-MNUA2, Olympus) for Alexa-350, BP470-490 BA510-550 (U-MNIBA2, Olympus) for Alexa-488, BP541-551 BA565-595 (XF37, Omega) for Alexa-555, and a longpass filter set BP520-550, BA580 (U-MWIG2) for Mitotracker CMX-Ros and chloroplast autofluorescence, and using a Hg arc lamp as a source of excitation light. Images were collected using a three charge-coupled device (3CCD) camera system, C7780-10 (Hamamatsu Photonics; Tokyo, Japan) and processed using the M8458-03 RCA-3CCD Photoshop plug-in software. For the photostability experiments, excitation light was irradiated through a U-MWIG2 filter set and each of the images through the U-MWIG2 and XF37 was captured successively less than 10 sec after the time indicated.


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Characterization of Antiserum Against C. merolae EF-Tu(mt)
A truncated recombinant protein containing amino acid residues 45–453 from EF-Tu and a six-histidine tag at the N terminus was expressed in E. coli The protein was purified and used to generate antibodies against EF-Tu in rats. Immunoblotting analysis showed that the raised antiserum specifically detected a 44-kD band from the total cell lysate of C. merolae (Figure 1A) . To localize EF-Tu, immunofluorescence microscopy was performed on C. merolae whose mitochondria were visualized by moderate staining with the fixable mitochondrial dye Mitotracker CMX-Ros. Because the mitochondrion and chloroplast are adjacent to each other in C. merolae, chloroplast autofluorescence can be distinguished from Mitotracker under phase contrast by its solid image (Figures 2A and 2B) . The antiserum specifically detected consistent signals that overlapped with the Mitotracker signal from both normal and dividing cells (Figures 2C and 2D).



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Figure 1

Immunoblotting analysis with the three antisera. Numbers at right indicate the molecular weight (kD). Dnm1, Anti-CmDnm1; FtsZ, Anti-CmFtsZ1; EF-Tu, Anti-EF-Tu.

 


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Figures 2–5

Figure 2 Indirect immunofluorescence with anti-EF-Tu antiserum. (A) Corresponding phase-contrast images of fixed and immunolabeled dividing (upper) and non-dividing (lower) cells. Chloroplasts are represented by bright and solid spots. (B) Fluorescence of Mitotracker-stained mitochondria and chloroplasts through a wide longpass filter set U-MWIG2. Chloroplasts can be distinguished from mitochondria in phase-contrast images. (C) EF-Tu localization detected by an FITC-conjugated secondary antibody. (D) Merged image of B and C. Bar = 1 µm.

Figure 3 Effects of different filter sets on captured images and photostability of chloroplasts and Alexa-555-labeled mitochondria. Captured images for the same field of view are aligned. Irradiation time is indicated above. (A) Images captured through the wide longpass filter set U-MWIG2. (B) Images captured through the narrow bandpass filter-set XF37. Bar = 1 µm.

Figure 4 Triple immunolabeling of CmFtsZ1, CmDnm1, and EF-Tu. CmDnm1 (Alexa-350), CmFtsZ (Alexa-488), and EF-Tu (Alexa-555) signals are shown in blue, green, and red, respectively. Panels showing individual or merged signals or images from the same cell are aligned horizontally. Panels are ordered vertically in the order of progression of cell division (A to D). (A) A cell containing an expanded chloroplast in preparation for division. (B) A cell with a constricted chloroplast. (C) A cell in which the chloroplast has divided and division of the mitochondrion is in process. (D) A cell finishing cytokinesis. P.C., phase-contrast image; FtsZ, Anti-CmFtsZ1; EF-Tu, Anti-EF-Tu; Dnm1, Anti-CmDnm1; Z1/Tu, merging FtsZ and EF-Tu; Z1/D1, merging FtsZ and Dnm1; D1/Tu, merging D1 and EF-Tu. Bar = 1 µm.

Figure 5 The cell, labeled as in Figure 4, from a different viewing angle. (A) Phase-contrast image. (B) Anti-CmFtsZ signal detected using Alexa-488. (C) Anti-CmDnm1 signal detected using Alexa-350. (D) Merged image of B and C. (E) Anti-EF-Tu signal detected using Alexa-555. (F) Merged image of B and E. (G) Merged image of C and E. (H) Merged image of B, C, and E. Bar = 1 µm.

 
Selection of Filter Set and Elimination of Chloroplast Autofluorescence
Because the most practical and suitable color combination for multifluorescent labeling is red and green, we devised a method of efficiently distinguishing the "red" autofluorescence of the chloroplast from the "red" signal of interest. Although the fluorescence of the chloroplast and certain red or orange dyes were distinguishable when directly observed (not shown), images captured through the 3CCD camera system did not have good contrast between these colors (Figure 3A). Several filter sets were examined for their ability to eliminate the chloroplast autofluorescence while passing certain signals, and the filter set XF37 (see Materials and Methods) was found to be effective. This filter eliminated most of the autofluorescence from both living and fixed chloroplasts of C. merolae while allowing sufficient passage of the orange signal from Alexa-555 (Figure 3B). Alternatively, it was possible to extinguish the autofluorescence of the chloroplast by photobleaching because the fluorescent dye was much more photostable than the chloroplast fluorescence. In the initial image, we could hardly distinguish the chloroplast and mitochondrial EF-Tu labeled with Alexa-555 when the image was reproduced through the 3CCD camera system (Figure 3A). After full irradiation with the excitation light for 1 min, the autofluorescence of the chloroplast was so decreased compared with the Alexa-555 signal that the labeled mitochondria were distinct (Figure 3A, 1min). Although the efficiency of elimination depends on the experimental conditions, the autofluorescence weakened further after 2 min of exposure to the excitation light (Figure 3A, 2 min).

Localization of FtsZ and Dynamin
Encouraged by the success of these methods, we performed a triple immunolabeling experiment to determine how the proteins of the inner and outer mitochondrial division machinery localized. Previously, the FtsZ and dynamin proteins in C. merolae (CmFtsZ1 and CmDnm1, respectively) had independently been shown to appear at the division site from the early and late stages of division, respectively, suggesting that each protein plays a different role (Nishida et al. 2003Go). In this study, both proteins were simultaneously labeled with an alternative mitochondrial marker, EF-Tu. During the early stage of division when the mitochondrion was not yet constricted, FtsZ appeared at the center of the mitochondrion (Figure 4A, FtsZ and Z1/Tu). Dispersed signals from FtsZ on the mitochondrion were also observed, consistent with observations in slime mold (Gilson et al. 2003Go). In the same cell, signals for dynamin were identified in several patches in the cytoplasm (Figure 4A, Dnm1), some of which seemed to be associated with the mitochondrion (Figure 4A, Tu/D1) and FtsZ (Figure 4A, Z1/D), although most of the patches were not. In a cell with a constricted mitochondrion (Figure 4B, EF-Tu), FtsZ was found only at the point of constriction, with a small amount along the edge of the mitochondrion (Figure 4B, Z1/Tu). Dynamin surrounded the FtsZ molecules at the site of constriction (Figure 4B, Dnm1, Z1/D1, and Tu/D1). In a cell whose mitochondrion had just divided (Figure 4C, EF-Tu), the FtsZ molecules were separated into the two daughter mitochondria and had a less intense signal (Figure 4C, FtsZ and Z1/Tu). On the other hand, dynamin assembled at the division site between the FtsZ patches (Figure 4C, Z1/D). When two daughter mitochondria had completely separated (Figure 4D, EF-Tu), dynamin was stuck to one side of the mitochondria, co-localizing with one of the two FtsZ patches (Figure 4D, Z1/D). Because the mitochondria in C. merolae are flat and round and are found between the chloroplast and the cytoplasmic region, we usually observed the mitochondria lying horizontally. However, a somewhat crooked cell gave us another viewpoint (Figure 5). In this cell, we could observe an apparently constricted mitochondrion vertically (Figure 5E), with a FtsZ belt crossing the center of the mitochondrion (Figures 5B and 5F) and several patches of dynamin associating on the side of the continuous FtsZ belt, which did not necessarily overlap (Figures 5C, 5D, 5G, and 5H).


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EF-Tu(mt) was shown to localize to both dividing and non-dividing mitochondria in C. merolae. Because the localization signal was identical to the signal seen in whole mitochondria stained with Mitotracker, the anti-EF-Tu antibody shows promise as a morphological mitochondrial marker because the protein is required for mitochondrial translation, is not implicated in a metabolic pathway, and is conserved among species. It is also thought to be present in large amounts and to be soluble and dispersed throughout the matrix.

Multiple fluorescent labeling is a direct and powerful method for examining localization or associations between cell components. Green fluorescent protein (GFP) (Chalfie et al. 1994Go) has enabled many cellular localization studies even within living cells. Nevertheless, immunofluorescent techniques still have a place in such studies since immunodetection methods must be used to confirm GFP localization studies because of the potential risk for artificial localization of the GFP fusion protein. In addition, there are still several organisms in which routine gene transformation is not practical.

For multiple immunofluorescence studies, the primary colors green and red are usually favored. However, the chloroplast of C. merolae occupies more than half of the cell and autofluorescences red, thereby making use of red fluorescence difficult if one is not interested in the chloroplast. In the previous study this problem was dealt with by analyzing images for differences in spectral characteristics between Mitotracker CMX-Ros and chloroplast autofluorescence, where the chloroplast autofluorescence had been decreased, but not completely eliminated, by methanol treatment. Image processing was then used to eliminate remaining traces of fluorescence. This method requires intense staining with Mitotracker, and even when a sample stains well it can be difficult to distinguish the mitochondria from the chloroplast when their images overlap. In addition, the efficiency of staining with Mitotracker depends on the condition of cells to be stained, and because of its ability to crosslink proteins, excess staining may cause mitochondrial disorder. Furthermore, recent studies reported that, in D. discoideum, Mitotracker accumulates in an electron-dense mass in mitochondria (van Es et al. 2001Go), which has been called the submitochondrial body (SMB) (Gilson et al. 2003Go). The SMB has been shown to contain TorA, which is required for directional responses in chemotactic gradients (van Es et al. 2001Go) and FtzB, one of the two FtsZ proteins in D. discoideum (Gilson et al. 2003Go), at least when they are expressed as GFP fusion proteins. Although no SMB has been found in C. merolae mitochondria, we noted that excess staining with Mitotracker caused altered mitochondrial morphology, some of which might have resulted from failure in constriction formation (unpublished data), although this may be a secondary effect. In any case, further studies are required, and the marker and method developed in this study would be useful for such studies.

The co-localization study of FtsZ and dynamin indicated that their localizations are indirectly related. We speculate that the machinery involved in mitochondrial division in C. merolae is as follows. First, an FtsZ ring forms at the center of the mitochondrion, determining the future division site, because FtsZ localizes before mitochondrial constriction occurs (Nishida et al. 2003Go; Figure 4A). This placement of the future division site is then transmitted through inner and outer membranes by unknown mechanisms. Following the signal, the outer MD ring forms along the same plane as the FtsZ ring at the inner membrane. The MD ring begins to constrict and recruits dynamin (Figure 5). This dynamin association is temporal because dynamin can tightly associate with the mitochondrion only as a continuous ring. Once formed, the dynamin ring increases in thickness by recruiting the remaining dynamin patches before final severing of the outer membrane. At this time, fission of the outer and inner membranes is not closely coupled, as seen by the different localizations of dynamin and FtsZ (Figures 4B and 4C). FtsZ then splits between the two daughter mitochondria, as seen by the weakened signals, suggesting that the FtsZ molecules are not tightly bound to one another at this stage but instead are associated with the membrane at the fission site.

Although FtsZ homologues are absent from the mitochondria in higher eukaryotes and no observable MD ring has been found in any organism other than C. merolae, dynamin-independent mitochondrial constriction has also been shown in S. cerevisiae (Legesse-Miller et al. 2003Go). Therefore, the mitochondrial constriction occurs by an unknown mechanism that remains to be discovered.


    Acknowledgments
 
Supported by Grants-in-Aid for Scientific Research on Priority Areas "Genome," by Grants 12446222 and 1287411 to TK, by a Grant-in-Aid for JSPS Fellows 11905 to KN from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by a Grant-in-Aid from the Promotion of Basic Research Activities for Innovative Biosciences (ProBRAIN) to TK.


    Footnotes
 
Received for publication March 17, 2004; accepted April 14, 2004


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 Summary
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 Materials and Methods
 Results
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 Literature Cited
 

Arimura S, Tsutsumi N (2002) A dynamin-like protein (ADL2b), rather than FtsZ, is involved in Arabidopsis mitochondrial division. Proc Natl Acad Sci USA 99:5727–5331[Abstract/Free Full Text]

Beech PL, Nheu T, Schultz T, Herbert S, Lithgow T, Gilson PR, McFadden GI (2000) Mitochondrial FtsZ in a chromophyte alga. Science 287:1276–1279[Abstract/Free Full Text]

Bleazard W, McCaffery JM, King EJ, Bale S, Mozdy A, Tieu Q, Nunnari J, et al. (1999) The dynamin-related GTPase Dnm1 regulates mitochondrial fission in yeast. Nature Cell Biol 1:298–304[CrossRef][Medline]

Bossy-Wetzel E, Barsoum MJ, Godzik A, Schwarzenbacher R, Lipton SA (2003) Mitochondrial fission in apoptosis, neurodegeneration and aging. Curr Opin Cell Biol 15:706–716[CrossRef][Medline]

Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263:802–805[Medline]

Gao H, Kadirjan-Kalbach D, Froehlich JE, Osteryoung KW (2003) ARC5, a cytosolic dynamin-like protein from plants, is part of the chloroplast division machinery. Proc Natl Acad Sci USA 100:4328–4333[Abstract/Free Full Text]

Gilson PR, Yu XC, Hereld D, Barth C, Savage A, Kiefel BR, Lay S, et al. (2003) Two Dictyostelium orthologs of the prokaryotic cell division protein FtsZ localize to mitochondria and are required for the maintenance of normal mitochondrial morphology. Eukaryot Cell 2:1315–1326[Abstract/Free Full Text]

Kuroiwa T, Kuroiwa H, Sakai A, Takahashi H, Toda K, Itoh R (1998) The division apparatus of plastids and mitochondria. Int Rev Cytol 181:1–41[Medline]

Labrousse AM, Zappaterra MD, Rube DA, van der Bliek AM (1999) C. elegans dynamin-related protein DRP-1 controls severing of the mitochondrial outer membrane. Mol Cell 4:815–826[Medline]

Legesse-Miller A, Massol RH, Kirchhausen T (2003) Constriction and Dnm1p recruitment are distinct processes in mitochondrial fission. Mol Biol Cell 14:1953–1963[Abstract/Free Full Text]

Margolin W (2003) Bacterial division: the fellowship of the ring. Curr Biol 13:R16–18[CrossRef][Medline]

Matsuzaki M, Misumi O Shin-I T, Maruyama S, Takahara M, Miyagishima S, Mori T, et al. (2004) Genome sequence of the ultra-small unicellular red alga Cyanidioschyzon merolae 10D. Nature 428:653–657[CrossRef][Medline]

Miyagishima SY, Nishida K, Kuroiwa T (2003) An evolutionary puzzle: chloroplast and mitochondrial division rings. Trends Plant Sci 8:432–438[CrossRef][Medline]

Morgan GW, Goulding D, Field MC (2003) The single dynamin-like protein of trypanosoma brucei regulates mitochondrial division and is not required for endocytosis. J Biol Chem (in press)

Mori T, Kuroiwa H, Takahara M, Miyagishima SY, Kuroiwa T (2001) Visualization of an FtsZ ring in chloroplasts of Lilium longiflorum leaves. Plant Cell Physiol 42:555–559[Abstract/Free Full Text]

Nishida K, Takahara M, Miyagishima SY, Kuroiwa H, Matsuzaki M, Kuroiwa T (2003) Dynamic recruitment of dynamin for final mitochondrial severance in a primitive red alga. Proc Natl Acad Sci USA 100:2146–2151[Abstract/Free Full Text]

Osteryoung KW, Nunnari J (2003) The division of endosymbiotic organelles. Science 302:1698–1704[Abstract/Free Full Text]

Osteryoung KW, Stokes KD, Rutherford SM, Percival AL, Lee WY (1998) Chloroplast division in higher plants requires members of two functionally divergent gene families with homology to bacterial ftsZ. Plant Cell 10:1991–2004[Abstract/Free Full Text]

Osteryoung KW, Vierling E (1995) Conserved cell and organelle division. Nature 376:473–474[Medline]

Otsuga D, Keegan BR, Brisch E, Thatcher JW, Hermann GJ, Bleazard W, Shaw JM (1998) The dynamin-related GTPase, Dnm1p, controls mitochondrial morphology in yeast. J Cell Biol 143:333–349[Abstract/Free Full Text]

Smirnova E, Shurland DL, Ryazantsev SN, van der Bliek AM (1998) A human dynamin-related protein controls the distribution of mitochondria. J Cell Biol 143:351–358[Abstract/Free Full Text]

Suzuki K, Ehara T, Osafune T, Kuroiwa H, Kawano S, Kuroiwa T (1994) Behavior of mitochondria, chloroplasts and their nuclei during the mitotic cycle in the ultramicroalga Cyanidioschyzon merolae. Eur J Cell Biol 63:280–288[Medline]

Takahara M, Takahashi H, Matsunaga S, Miyagishima S, Takano H, Sakai A, Kawano S, et al. (2000) A putative mitochondrial ftsZ gene is present in the unicellular primitive red alga Cyanidioschyzon merolae. Mol Gene Genet 264:452–460[CrossRef]

van der Bliek AM (1999) Functional diversity in the dynamin family. Trends Cell Biol 9:96–102[CrossRef][Medline]

van Es S, Wessels D, Soll DR, Borleis J, Devreotes PN (2001) Tortoise, a novel mitochondrial protein, is required for directional responses of Dictyostelium in chemotactic gradients. J Cell Biol 152:621–632[Abstract/Free Full Text]

Vitha S, McAndrew RS, Osteryoung KW (2001) FtsZ ring formation at the chloroplast division site in plants. J Cell Biol 153:111–120[Abstract/Free Full Text]

Yaffe MP (1999) Dynamic mitochondria. Nat Cell Biol 1:E149–150.[CrossRef][Medline]