RAPID COMMUNICATION |
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
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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Summary |
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Key Words: FtsZ dynamin elongation factor Tu mitochondrial division Cyanidioschyzon merolae Alexa triple immunofluorescence
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Introduction |
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FtsZ is a self-assembling GTPase originally involved in bacterial cell division (reviewed by Margolin 2003), which was imported via endosymbiosis for use in the chloroplast (Osteryoung and Vierling 1995
), and some primitive mitochondria (Beech et al. 2000
; Takahara et al. 2000
). FtsZs are required for normal division of the chloroplast (Osteryoung et al. 1998
) or mitochondrion (Gilson et al. 2003
) and form rings around the future division sites of chloroplasts (Mori et al. 2001
; Vitha et al. 2001
) and mitochondria (Beech et al. 2000
; Nishida et al. 2003
) 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 1999). The involvement of dynamin in mitochondrial fission was first characterized in budding yeast as the protein Dnm1p (Otsuga et al. 1998
; Bleazard et al. 1999
) and was later found to be widespread in eukaryotes: humans (Smirnova et al. 1998
), nematodes (Labrousse et al. 1999
), higher plants (Arimura and Tsutsumi 2002
), red algae (Nishida et al. 2003
), and parasites (Morgan et al. 2003
). These two dynamin homologues found in the C. merolae genome (Matsuzaki et al. 2004
) have been characterized as dynamins involved in mitochondrial division (Nishida et al. 2003
) and chloroplast division (Miyagishima et al. 2003
). Arabidopsis thaliana also uses a dynamin for chloroplast division (Gao et al. 2003
). In Trypanosoma brucei, only one dynamin, implicated in mitochondrial division but not involved in endocytosis, was found (Morgan et al. 2003
). These findings further support the idea that dynamin in eukaryotes was originally involved in organelle division (Nishida et al. 2003
). Dynamins involved in mitochondrial division are associated with the cytosolic surface of the outer membrane (Otsuga et al. 1998
) and are involved in the fission of the outer but not the inner membrane (Labrousse et al. 1999
). 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. 2000
), Mallomonas splendens (Beech et al. 2000
), and Dictyostelium discoideum (Gilson et al. 2003
).
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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Materials and Methods |
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Cell Preparation
C. merolae cells were grown and synchronized as previously described (Suzuki et al. 1994). Cell fixation was performed as previously described (Nishida et al. 2003
) 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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Results |
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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. 2003). 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. 2003
). 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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Discussion |
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Multiple fluorescent labeling is a direct and powerful method for examining localization or associations between cell components. Green fluorescent protein (GFP) (Chalfie et al. 1994) 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. 2001), which has been called the submitochondrial body (SMB) (Gilson et al. 2003
). The SMB has been shown to contain TorA, which is required for directional responses in chemotactic gradients (van Es et al. 2001
) and FtzB, one of the two FtsZ proteins in D. discoideum (Gilson et al. 2003
), 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. 2003; 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. 2003). Therefore, the mitochondrial constriction occurs by an unknown mechanism that remains to be discovered.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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Arimura S, Tsutsumi N (2002) A dynamin-like protein (ADL2b), rather than FtsZ, is involved in Arabidopsis mitochondrial division. Proc Natl Acad Sci USA 99:57275331
Beech PL, Nheu T, Schultz T, Herbert S, Lithgow T, Gilson PR, McFadden GI (2000) Mitochondrial FtsZ in a chromophyte alga. Science 287:12761279
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 1:298304[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 15:706716[CrossRef][Medline]
Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263:802805[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 100:43284333
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 2:13151326
Kuroiwa T, Kuroiwa H, Sakai A, Takahashi H, Toda K, Itoh R (1998) The division apparatus of plastids and mitochondria. Int Rev Cytol 181:141[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 4:815826[Medline]
Legesse-Miller A, Massol RH, Kirchhausen T (2003) Constriction and Dnm1p recruitment are distinct processes in mitochondrial fission. Mol Biol Cell 14:19531963
Margolin W (2003) Bacterial division: the fellowship of the ring. Curr Biol 13:R1618[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 428:653657[CrossRef][Medline]
Miyagishima SY, Nishida K, Kuroiwa T (2003) An evolutionary puzzle: chloroplast and mitochondrial division rings. Trends Plant Sci 8:432438[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 42:555559
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 100:21462151
Osteryoung KW, Nunnari J (2003) The division of endosymbiotic organelles. Science 302:16981704
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 10:19912004
Osteryoung KW, Vierling E (1995) Conserved cell and organelle division. Nature 376:473474[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 143:333349
Smirnova E, Shurland DL, Ryazantsev SN, van der Bliek AM (1998) A human dynamin-related protein controls the distribution of mitochondria. J Cell Biol 143:351358
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 63:280288[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 264:452460[CrossRef]
van der Bliek AM (1999) Functional diversity in the dynamin family. Trends Cell Biol 9:96102[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 152:621632
Vitha S, McAndrew RS, Osteryoung KW (2001) FtsZ ring formation at the chloroplast division site in plants. J Cell Biol 153:111120
Yaffe MP (1999) Dynamic mitochondria. Nat Cell Biol 1:E149150.[CrossRef][Medline]