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Keywords:

  • ascidian;
  • dual distribution;
  • ectopic ATP synthase;
  • mitochondria;
  • myoplasm

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Previously, we revealed that p58, one of the ascidian maternal factors, is identical to the alpha-subunit of F1-ATP synthase (ATPα), a protein complex of the inner mitochondrial membrane. In the current study, we used immunological probes for ascidian mitochondria components to show that the ascidian ATPα is ectopically localized to the cytosol. Virtually all mitochondrial components were localized to the mitochondria-rich myoplasm. However, in detail, ATP synthase subunits and the matrix proteins showed different localization patterns. At least at the crescent stage, transmission electron microscopy (TEM) distinguished the mitochondria-less, endoplasmic reticulum (ER)-rich cortical region and the mitochondria-rich internal region. ATPα was enriched in the cortical region and MnSOD was limited to the internal region. Using subcellular fractionation, although all of the mitochondria components were highly enriched in the mitochondria-enriched fraction, a considerable amount of ATPα and F1-ATP synthase beta-subunit (ATPβ) were recovered in the insoluble cytoplasmic fraction. Even under these conditions, F1-ATP synthase gamma-subunit (ATPγ) and F0-ATP synthase subunit b (ATPb) were not recovered in the insoluble cytoplasmic fraction. This result strongly supports the exomitochondrial localization of both ATPα and ATPβ. In addition, the detergent extraction of eggs supports the idea that these cytosolic ATP synthase subunits are associated with the egg cytoskeleton. These results suggest that the subunits of ATP synthase might play dual roles at different subcellular compartments during early development.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Conklin closely observed the cell lineage and the egg organization of Cynthia (Styela) partita, and described the “yellow crescent” at the posterior pole just before the first cleavage event (Conklin 1905a). As this yellow cytoplasm was distributed among all larval muscle cells, he proposed to designate the cytoplasmic region as “myoplasm” (Conklin 1905b). Until the 1990s, the myoplasm was recognized as a cytoplasmic region containing pre-localized egg cytoplasmic information responsible for specifying larval tail-muscle cells (reviewed by Satoh et al. 1990; Satoh 1994).

When the maternal macho-1 mRNA, ascidian muscle determinant, was discovered at the end of the last century (Nishida & Sawada 2001), the cortical region of the myoplasm, where macho-1 mRNA is localized, was thought to be the sole location of maternal information. Moreover, many maternal mRNAs, so-called “type-I postplasmic RNAs” (Makabe et al. 2001), or “postplasmic/PEM RNAs” (Prodon et al. 2005) are also localized to the cortical region. These findings pointed out the importance of the cortical region of the myoplasm.

Ultrastructurally, myoplasm is composed of pigment granules, an aggregation of mitochondria, endoplasmic reticula, fine granular materials, and a cytoskeletal framework (Berg & Humphreys 1960; Jeffery & Meier 1983). Speksnijder et al. (1993) revealed the endoplasmic reticulum (ER) network within the ascidian egg cytoplasm and the stuck of ER in the myoplasm. Gualtieri & Sardet (1989) revealed the cortical ER (cER) domain in the cortex of the myoplasm by the techniques of confocal microscopy and isolated cortices. High-resolution confocal microscopy and in situ hybridization revealed the co-localization of postplasmic mRNA and the cER (Sardet et al. 2003, 2005; Prodon et al. 2005).

In previous studies, p58 was identified as an ascidian maternal factor, localized throughout the entire myoplasm and having a role in muscle differentiation. Swalla et al. (1991) first reported p58, which was recognized by the NN18 antibody, an anti-neurofilament 160 monoclonal antibody. They also reported that p58 is necessary for restoration of larval features in M. occulta × M. oculata hybrids and also proposed p58 as a cytoskeletal component (Jeffery & Swalla 1992). On the other hand, myoplasmin-C1 was one of the myoplasm-specific antigens recognized by monoclonal antibodies raised against isolated Ciona whole myoplasm, and was suggested to have a role in muscle differentiation (Nishikata et al. 1987). The myoplasmin-C1 has heptad-repeat domains and is suggested to form homo- or hetero-oligomeric complexes in vivo through an alpha-helix coiled-coil structure (Nishikata & Wada 1996). Actually, myoplasmin-C1 is detergent insoluble and pull-down experiments suggested direct binding to p58 (Nishikata & Wada 1996). The functions of these proteins and the importance of the internal region of the myoplasm have yet to be elucidated. However, recently, using cDNA-library screening with NN18 antibody and amino acid sequencing of the immunoprecipitated p58, we found that p58 is identical to the F1-ATP synthase alpha-subunit (ATPα; genebank accession number AB071977; data not shown). Although, much more research is required to further define the molecular nature of ascidian ATPα, mitochondrial involvement in ascidian early development becomes an interesting theme.

In this study, we prepared immunological probes against Ciona mitochondrial components and carried out immunohistological double staining and Western blotting with subcellular fractionation. The comprehensive double staining revealed different distribution patterns between two types of mitochondrial proteins; F1-ATP synthase subunits, α and β (ATPβ), and matrix proteins, MnSOD and PDHE1α. These four antigens were localized to the mitochondria-rich region of the myoplasm. However, during cytoplasmic and cortical rearrangement, ATPα and ATPβ became enriched in the mitochondria-less cortical region of the myoplasm, the so-called cER-mRNA domain. By Western blotting, ATPα and β were recovered in the insoluble-cytoplasmic fraction along with the mitochondria-enriched fraction, while three matrix components (MnSOD, PDHE1α, and HSP60), F1-ATP synthase gamma-subunit (ATPγ), and F0-ATP synthase subunit b (ATPb) were recovered only in the mitochondria-enriched fraction. From these data, we conclude that ATPα and ATPβ are localized exomitochondrially to the cortical ER-rich domain of the myoplasm.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Animals, gonads, and embryos

Wild type Ciona intestinalis adults were provided by Y. Satou (Kyoto University) and M. Yoshida (University of Tokyo) by the National Bio-Resource Project of MEXT, Japan. The animals were kept at 18˚C in laboratory tanks with a closed water circulation system. Gonads and eggs were obtained surgically. Eggs were inseminated with a diluted suspension of non-self sperm. The embryos were reared in Millipore-filtered (pore size = 0.45 μm) seawater at 18°C.

Antibodies

To investigate the localization of entire F0F1-ATP synthase, we generated antibodies against Ciona F1-ATP synthase alpha-subunit (anti-α), beta-subunit (anti-β), gamma-subunit (anti-ATPγ), and against Ciona F0-ATP synthase subunit b (anti-ATPb). These antibodies were generated by MBL, Nagoya, Japan (anti-α and β) and Hokkaido System Science, Sapporo, Japan (anti-ATPγ and ATPb) with the following oligopeptides as antigens designed from the Ciona genome database: CILDSIRNEGKITESTEA (KH.C10.579) for anti-α; CPIDERGPVDTEHFAGIH (KH.C3.87) for anti-β;SKASVYVPQLVQVRC and CPFFSVEVINQSPTI (KH.C14.125) for anti-ATPγ; RILFKAGRPVC and CVQKVDSQLAHGHMV (KH.C1.670) for anti-ATPb.

In this report, NN18 mouse monoclonal antibody (Sigma, St Louis, MO, USA N5264) was used as the anti-F1-ATP synthase alpha-subunit amtibody (anti-ATPα antibody). The specificity of NN18 antibody against Ciona proteins was confirmed by following two experiments (data not shown). First, we screened a Ciona-cDNA expression library with NN18 antibody. All positive clones encoded the same protein, F1-ATP synthase alpha-subunit “CiATPsynA” (Genbank accession number AB071977). Second, we carried out the immunoprecipitation with NN18 antibody from Ciona-gonad whole lysate and determined the N-terminal amino acid sequence of the precipitated protein. The resulting sequence of 27 amino acid residues was almost identical (26 out of 27) to the N-terminal amino acid sequence of the matured form of Ciona F1-ATP synthase alpha-subunit (KH.C10.579).

Immunohistochemistry

Ciona sperm was fixed on the cover slip with 100% methanol, followed by 100% ethanol. Gonads and embryos were fixed in the same way, embedded in Polyester Wax (Steedman 1957; BDH, Poole, UK) and sectioned at 8 μm. After removal of the Polyester Wax, the sectioned specimens were stained with appropriate sets of primary and secondary antibodies from among the following: anti-ATPα antibody (1:200 dilution), anti-ATP5B chicken antisera (anti-ATPβ antibody; Sigma, GW22827; 1:1000 dilution), anti-MnSOD rabbit antisera (StressMarq Biosciences, Victoria, Canada, SPC-117C/D; 1:40 dilution), anti-PDHE1α mouse monoclonal antibody (MitoScience, Eugene, OR, MSP07; 1:50 dilution), anti-aPKC rabbit antisera (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-216; 1:200 dilution), anti-kinesin monoclonal antibody (BioMakor, Rehovot, Israel, 6508; 1:2000 dilution), rabbit anti-chicken IgG (Sigma, C-6778; 1:200 dilution), rhodamine-conjugated goat anti-mouse IgG+M (BioSource International, Camarillo, CA, USA, AMI0706; 1:200 dilution), fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (American Qualex Antibodies, San Clemente, CA, USA, A102FS; 1:200 dilution), FITC-conjugated goat anti-mouse IgG+M (American Qualex Antibodies, A108FS; 1:200 dilution), Alexa Fluor 568-conjugated goat anti-chicken IgG (Molecular Probes, Eugene, OR, USA, A11041; 1:2000 dilution). The stained specimens were observed using a BioRevo BZ-900 (Keyence, Osaka, Japan) or LSM700 confocal microscope (Carl Zeiss, Jena, Germany).

Transmission electron microscope observation

Ciona fertilized eggs were fixed in 2.0% paraformaldehyde, 3.0% glutaraldehyde and 3.0% NaCl in 0.2 mol/L sodium phosphate buffer (pH 7.4) for 2 h at room temperature, followed by several rinses in buffer. The samples were postfixed in 1.0% osmium tetroxide in the same buffer for 30 min. The samples were then rinsed in distilled water and stained with 1.0% uranyl acetate for 1h. After rinsing in distilled water, the samples were dehydrated in acetone and embedded in Spurr resin (Spurr 1969). Ultrathin sections were cut and stained with lead citrate for 10 min. The sections were observed under an electron microscope, JEM-1400 (JEOL, Tokyo, Japan). The numbers of mitochondria were counted manually from the photomicrographs.

Subcellular fractionation of Ciona gonad homogenate

Gonads were washed several times with ice-cold homogenization solution (HS; 0.5 m sucrose, 50 mm Tris–HCl pH 7.5, 20 mm MgCl2, 10 mm Ethylene Glycol Bis(β-aminoethylether)-N,N,N',N'-tetraacetic Acid [EGTA]). This HS was modified from the previously described method (Fujiwara & Yasumasu 1997). The washed gonads were suspended in 10 volumes of ice-cold HS and homogenized with a Potter-Elvehjem glass homogenizer fitted with a Teflon pestle for 10 strokes. The homogenate was filtered through a nylon mesh (N-NO.270T; NBC, Tokyo, Japan) and centrifuged at 500 g for 10 min. The homogenate was stratified into a pellet, a supernatant, and a fluffy layer. The pellet and the fluffy layer were designated the insoluble cytoplasmic mass (IC) fraction and fluffy layer (FL) fraction, respectively. The supernatant was centrifuged at 10 000 g for 10 min and the resulting pellet and supernatant were designated the mitochondria-enriched (ME) fraction and soluble cytoplasmic (SC) fraction, respectively. The concentration of these protein samples was determined by the Bicinchoninic Acid assay using Bicinchoninic Acid Solution (Sigma, B9643) and Copper (II) Sulfate Solution (Sigma, C2284). The same amount of the total protein was applied in each subsequent experiment.

Preparation of extracted eggs

The method for the preparation of extracted eggs was modified from the previously described method (Nishikata & Wada 1996). Unfertilized Ciona eggs were washed three times with Ca2+-, Mg2+-free seawater containing 1 mm EDTA. The washed eggs were treated with extraction buffer (20 mm MgCl2, 10 mm KCl, 10 mm EGTA, 2% Triton X-100, 20% glycerol, 25 mm imidazole, pH6.9). The extracted eggs were collected by centrifugation and used for immunohistochemistry and Western blotting.

SDS–PAGE and Western blot

Protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and blotted onto polyvinylidene fluoride (PVDF) membrane (Immobilon, Millipore, Billerica, MA, USA). Antibodies used in this study were as follows: anti-ATPα antibody (1:2000 dilution), anti-ATP5B chicken antisera (anti-ATPβ antibody; 1:16 000 dilution), anti-MnSOD rabbit antisera (1:800 dilution), anti-PDHE1α mouse monoclonal antibody (1:800 dilution), anti-GroEL rabbit antisera (Stressgen, Victoria, Canada, SPS-875; 1:800 dilution), anti-aPKC rabbit antisera (1:2000 dilution), anti-ATPγ rabbit antisera (1:800 dilution), anti-ATPb rabbit antisera (1:800 dilution), anti-β-Tubulin mouse monoclonal antibody (Sigma, T5293; 1:500 dilution), rabbit anti-chicken IgG (Sigma, C-6778; 1:1000 dilution), AP conjugated goat anti-mouse IgG+A+M (Zymed Laboratories, South San Francisco, CA, USA 62-6422; 1:5000 dilution), AP conjugated goat anti-rabbit IgG (American Qualex Antibodies, A102AT; 1:5000 dilution).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Quest for the immunological probes for the Ciona mitochondria

Mitochondria are specialized for their important roles in many biological events, such as apoptosis, aging, carcinogenesis, and disease pathogenesis (Svensson 2010). For this reason, many kinds of mitochondrial probes have recently become commercially available. We have selected some of these antibodies based on amino acid sequence homology between the immunogen used to generate the antibody and the equivalent protein in Ciona. In addition, we have raised some rabbit antisera against oligopeptides designed from the genomic sequence information of Ciona (Ghost database: http://ghost.zool.kyoto-u.ac.jp/cgi-bin/gb2/gbrowse/kh/). These candidate antibodies were first screened by Western blotting against Ciona-gonad total protein. The antibodies available for probing the Ciona mitochondria are summarized in Table 1. The antibodies recognized the proteins as a single band of predicted-molecular weight of each immunogen in Ciona (data not shown).

Table 1. Available immunological probes for Ciona mitochondria
NameTarget moleculeImmunogenFromApplicationaM.W.bLocalizationSource or reference
  1. a

    Applicable experimental methods: IC, immunohistochemistry; IP, immunoprecipitation; WB, Western blotting.

  2. b

    Molecular weight predicted from the Ciona genome sequence.

Anti-MnSODMnSOD (manganese superoxide dismutase)Rat MnSODRabbit antiseraWB, IC24 kDaMatrixStressMarqBiosciences, SPC-117C/D
Anti-PDHE1αPDHE1α (pyruvate dehydrogenase E1 component subunit alpha)Human PDHE1αMouse monoclonal antibodyWB, IC43 kDaMatrixMitoScience, MSP07
Anti-GroELHSP60 (60kDa heat shock protein)E. coli GroELRabbit antiseraWB61 kDaMatrix/cytosolStressgen, SPS-875
NN18 (anti-ATPα)ATPα (F1-ATP synthase alpha-subunit)Porcine neurofilament 160Mouse monoclonal antibodyWB, IC, IP60 kDaInner membraneSigma, N5264
Anti-αATPαOligopeptides designed from Ciona ATPα sequenceRabbit antiseraWB60 kDaInner membraneThis study
Anti-ATP5B (anti-ATPβ)ATPβ (F1-ATP synthase beta-subunit)Human ATPβChickin antiseraWB, IC56 kDaInner membraneSigma-Aldrich, GW22827
Anti-βATPβOligopeptides designed from Ciona ATPβ sequenceRabbit antiseraWB56 kDaInner membraneThis study
Anti-ATPγATPγ (F1-ATP synthase gamma-subunit)Oligopeptides designed from Ciona ATPγ sequenceRabbit antiseraWB33 kDaInner membraneThis study
Anti-ATPbATPb (F0-ATP synthase subunit b)Oligopeptides designed from Ciona ATPb sequenceRabbit antiseraWB30 kDaInner membraneThis study

For the second level of screening, we used immunofluorescent staining against Ciona sperm. Ciona sperm is a suitable material because each one has only a single mitochondrion adjacent to the sperm-head nucleus. Moreover, during penetration, sperm shows a characteristic behavior of the mitochondrion, the so-called sperm reaction (Lambert & Koch 1988). This process can occur in vitro, and sperm mitochondrion is translocated to the posterior tail tip and becomes spherical (Lambert & Epel 1979).

Fixed sperm were double-stained with anti-ATPα antibody and anti-ATPβ antibody or anti-MnSOD antibody, and counterstained with DAPI (4'6'-diamidino-2-phenylindole dihydrochloride). A mitochondrion at the side of the sperm-head nucleus showed positive staining with these antibodies and these signals nicely merged (Fig. 1). A mitochondrion was also identified in the tail of the sperm that induced the sperm reaction. (Fig. 1H). Similar double staining was carried out with anti-ATPβ and anti-PDHE1α antibodies. These antibodies also showed similar mitochondrion-specific staining (data not shown).

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Figure 1. Antibody staining of Ciona mitochondria. Ciona sperm were fixed and double-stained with two different antibodies and counter stained with DAPI (4´6´-diamidino-2-phenylindole dihydrochloride) (C, G; DNA; blue). Merged images (Merge) were shown in D and H. `(A–D) Comparison of anti-ATPα antibody (A; ATPα; Rhodamine, red) and anti-MnSOD antibody (B; MnSOD; FITC [fluorescein isothiocyanate], green). (E–H) Comparison of anti-ATPα antibody (E; ATPα; Rhodamine, red) and anti-ATPβ antibody staining (F; ATPβ; FITC, green). Arrow and arrowhead indicate a single mitochondrion and a nucleus of sperm that induced the sperm reaction, respectively. All three antibodies recognize sperm mitochondria similarly. Scale bar, 5 μm.

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Subcellular localization of ATP synthase before and after fertilization

The behavior of mitochondria during cytoplasmic and cortical rearrangement has been described in detail (e.g. Sardet et al. 2007). In order to compare the localization patterns of exomitochondrial ATP synthase and mitochondria, sectioned specimens of unfertilized eggs (Fig. 2A,B) and fertilized eggs at 5 min (Fig. 2C,D) and 45 min (Fig. 2E–G) after fertilization were double-stained with anti-ATPα as well as anti-MnSOD, anti-ATPβ or anti-PDHE1α antibodies.

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Figure 2. Subcellular localization of mitochondrial proteins in unfertilized and fertilized eggs. (A, B) Vertical section of unfertilized egg. (C, D) Vertical section of fertilized egg 5 min after fertilization. (E–G) Sagittal section of fertilized egg 45 min after fertilization, the so-called crescent stage. Animal pole is up. (A, C, E) Double staining with anti-ATPα antibody (ATPα; Rhodamine, red) and anti-MnSOD antibody (MnSOD; FITC [fluorescein isothiocyanate], green). (B, D, F) Double staining with anti-ATPα antibody (ATPα; Rhodamine, red) and anti-ATPβ antibody (ATPβ; FITC, green). (G) Double staining with anti-ATPβ antibody (ATPβ; Alexa Fluor 568, red) and anti-PDHE1α antibody (PDHE1α; FITC, green). Merged image (Merge) of each set is also shown. The areas indicated by the white rectangle were enlarged and the line profiles of fluorescent intensity (red line, Rhodamine or Alexa Fluor 568; green line, FITC) along the indicated white dotted line are shown. Scale bar, 50 μm and 10 μm (enlarged view).

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Throughout these stages, merged images of the double staining with anti-ATPα and anti-ATPβ antibodies showed a uniform yellowish pattern, suggesting the co-localization of these two antigens (Fig. 2B,D,F).

On the other hand, the double staining with anti-ATPα and anti-MnSOD antibodies showed a slight difference in staining pattern. In the unfertilized egg (Fig. 2A), both antibodies stained the mitochondria-rich cortical myoplasm. In the merged image, the cortical myoplasm showed a yellowish signal, but green- and red-colored small dot signals were found in the neighboring egg cytoplasm. In the egg 5 min after fertilization (Fig. 2C), although single color staining showed very similar staining patterns of the myoplasm, the merged image showed a clear difference. The 1–3 μm in thickness of the cortical myoplasm was intensely stained with anti-ATPα antibody, while weakly stained with anti-MnSOD antibody. In the egg 45 min after fertilization (Fig. 2E), different staining patterns for anti-ATPα and anti-MnSOD antibodies were obvious. The cortical region, which stained strongly for ATPα and weakly for MnSOD, was 2–6 μm in thickness. Double staining with anti-ATPβ and anti-PDHE1α antibodies yielded a very similar pattern as that with anti-ATPα and anti-MnSOD antibodies (Fig. 2G).

In order to clarify which antibody represented the mitochondria localization pattern, we carried out transmission electron microscopic (TEM) observation of the myoplasm at 45 min after fertilization. We found the mitochondria-less and ER-rich cortical region, which was about 2–6 μm in thickness beneath the plasma membrane (Fig. 3A,B). We also carefully compared the fluorescent-intensity of the immunofluorescent signal and the number of mitochondria from TEM observation. We took belt-like areas in the myoplasmic region along the anterior posterior axis, and counted the number of mitochondria in every 4 μm (Fig. 3B). The data revealed that the cytoplasm of the first 4 μm in thickness beneath the plasma membrane had fewer amounts of mitochondria. Then, re-calculation of the fluorescent intensity was performed, averaged every 4 μm (Fig. 3C). The cortical most 4 μm in thickness showed intense anti-ATPα antibody staining and weak anti-MnSOD antibody staining. Comparing these results, we concluded that the localization pattern of mitochondria was recapitulated by anti-MnSOD antibody staining. Moreover, the line profiles of double staining with anti-ATPβ and anti-PDHE1α antibodies (Fig. 2G) were almost identical to the anti-ATPα and anti-MnSOD antibodies staining, supporting the congruence between localization patterns of mitochondria and those of mitochondrial matrix components. Hence, the mitochondria-less and ER-rich cortical region of the myoplasm corresponded closely to the ATP-synthase rich cortical region of the myoplasm.

image

Figure 3. Comparison of transmission electron microscopy (TEM) and immunofluorescent staining. (A) A low magnification of the sagittal section of a crescent stage embryo. Dotted line and arrowheads indicate the myoplasmic region. (B) A higher magnification of myoplasmic region indicated in A. The number of mitochondria within the 3 × 32-μm rectangle were counted every 4-μm. The average of the data obtained from three different areas is indicated in the graph with standard deviation. (C) Same photo as in Figure 2E. Line profiles of red and green fluorescent intensity along the indicated white line was averaged every 4 μm. Scale bar, 20 μm in A.

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Subcellular localization of ATP synthase during oogenesis

We can distinguish five stages of oocyte development by staining their accessory cells with DAPI (Shimai et al. 2010). In stage 1 oocytes (10–60 μm in diameter), several undifferentiated accessory cells are scattered around the oocyte. In stage 2 oocytes (60–75 μm in diameter), accessory cells number is increased and they are stacked around the oocyte. In stage 3 oocytes (75–90 μm in diameter), the accessory cells have differentiated into test cells and the interspersed inner follicle cells and the chorion become obvious. In some cases, a perivitelline space is transiently observed. In stage 4 oocytes (90–100 μm in diameter), the cytoplasm of the oocyte has expanded and the clusters of test cells are buried in the oocyte cortex. This structure was designated the pocket structure by Shimai et al. (2010). In stage 5 oocytes (100–130 μm in diameter), the germinal vesicle has broken down, and the pocket structures have disappeared. The oocyte surface becomes smooth and an obvious perivitelline space has formed; this is the stage just before the unfertilized egg (Prodon et al. 2006).

In all stages of oogenesis, merged images of the double staining with anti-ATPα and anti-ATPβ antibodies essentially showed a uniform yellowish signal, suggesting that these two antigens are co-localized even in the gonad (Fig. 4I–M).

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Figure 4. Subcellular localization of mitochondrial proteins during oogenesis. Histological sections of the stage 1 (A–D, I), stage 2 (E, J), stage 3 (F, K), stage 4 (G, L), and stage 5 (H, M) oocytes were double-stained with anti-MnSOD antibody (A, B, D–H; MnSOD; FITC [fluorescein isothiocyanate], green) or anti-ATPα antibody (A–C, E–M; ATPα; Rhodamine, red), and anti-ATPβ antibody (I–M; ATPβ; FITC, green), counter stained with DAPI [4´6´-diamidino-2-phenylindole dihydrochloride] (A, E–M; DNA; blue). The area indicated by the white rectangle in A was enlarged and the staining patterns of anti-ATPα antibody (C) and anti-MnSOD antibody (D), and the merged image (B) are shown. Arrowheads show the pocket structure of the stage 4 oocytes (G, L). GV, germinal vesicle. Staging of oocyte was according to the criteria indicated by Shimai et al. (2010). Scale bars, 50 and 2 μm (enlarged view).

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On the other hand, anti-ATPα and anti-MnSOD antibody-staining patterns were distinct. In stage 1 oocytes (Fig. 4A–D), both anti-ATPα and anti-MnSOD antibodies stained a string-like structure of mitochondria. However, the merged image was not uniformly yellow, suggesting that ATPα and MnSOD have a different distribution within the mitochondria. In stage 2 oocyte (Fig. 4E), the string-like staining of the previous stage developed to a more punctate pattern. This change was more obvious with the anti-ATPα antibody (Fig. 4J). In stage 3 oocytes (Fig. 4F), the cytoplasm was filled with intense punctate staining for the anti-ATPα antibody, while the MnSOD staining became faint and its positive region was restricted to the relatively thick cortical area. In stage 4 oocytes (Fig. 4G), the staining region for the anti-ATPα antibody was restricted to the cortical region, which was adjacent to the pocket structure, and overlapped with the cortical staining of the anti-MnSOD antibody. However, the anti-MnSOD antibody signal grew faint, as the merged image shows, as if only the ATPα signal was localized to the cortical area. The staining pattern of stage 5 oocytes was very close to that of unfertilized egg (Fig. 4H). Although the animal pole of unfertilized egg was free of mitochondria, the entire cortex of the stage 5 oocyte stained positive with both antibodies.

Subcellular localization of ATP synthase during early development

During the cleavage stages, a unique structure called the CAB (centrosome-attracting body) is observed at the posterior pole (Hibino et al. 1998). It is suggested to have important roles for ascidian development such as in the asymmetric cleavages (Nishikata et al. 1999), embryonic axis formation (Negishi et al. 2007) and germ cell specification (Kumano et al. 2011; Shirae-Kurabayashi et al. 2011). TEM studies revealed that the CAB contains an electron-dense matrix, ER, and ribosomes, but no mitochondria (Iseto & Nishida 1999). Moreover, many postplasmic/PEM RNAs including macho-1 (Prodon et al. 2007) and vasa homologue (Shirae-Kurabayashi et al. 2006), and some interesting proteins including PEM (Shirae-Kurabayashi et al. 2011) and aPKC (Patalano et al. 2006) are localized to the CAB.

In order to find out whether the CAB contains the exomitochondrial ATP synthase, we carried out double staining with anti-ATPα and anti-aPKC antibodies or with anti-ATPβ and anti-kinesin antibodies. The anti-aPKC and anti-kinesin antibodies positively stain the CAB during the cleavage stages (Nishikata et al. 1999; Patalano et al. 2006). As shown in Figure 5, anti-aPKC and anti-kinesin antibodies stained the CAB. On the other hand, the anti-ATPα and anti-ATPβ antibodies did not stain the CAB but stained the adjacent cytoplasmic region.

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Figure 5. ATPα did not localize to the centrosome-attracting body (CAB) at the 16-cell stage. Horizontal sections of the posterior pole region of a 16-cell stage embryo were double-stained with anti-ATPα antibody (A; ATPα; Rhodamine, red), and anti-aPKC antibody (B; aPKC; FITC, green) or with anti-ATPα antibody (D; ATPβ; Alexa Fluor 568, red), and anti-kinesin antibody (E; Kinesin; FITC [fluorescein isothiocyanate], green). Merged images are also shown (C, F; Merge). Posterior is up. Arrows indicate the CAB. Scale bar, 10 μm.

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During the tail-bud stage in the embryo, some tissues are differentiated, and the content of mitochondria varies according to the tissue type. Muscle cells and neural tissues are rich in mitochondria. As shown in Figure 6D–F, the merged images of the double staining with anti-ATPα and anti-ATPβ antibodies reveal intense yellowish staining in muscle, mesenchyme, and brain, suggesting that these two antigens are co-localized even in the differentiated tissues. On the other hand, anti-ATPα and anti-MnSOD antibodies showed quite distinct staining patterns (Fig. 6A–C). The anti-MnSOD antibody stained the entire cytoplasm of muscle cells brightly as well as the small dots of epidermal cells, while anti-ATPα antibody stained muscle, mesenchyme, and brain cells. Even in the muscle cells, the merged image shows that the signal intensities of these two antibodies are different within the cell.

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Figure 6. Subcellular localization of mitochondrial proteins at the tail-bud stage. Horizontal sections of the tail-bud stage embryo were double-stained with anti-ATPα antibody (A, D; ATPα; Rhodamine, red), and anti-MnSOD antibody (B; MnSOD; FITC [fluorescein isothiocyanate], green) or anti-ATPβ antibody (E; ATPβ; FITC, green). Merged image of each set is also shown (C, F; Merge). Anterior is to the left. Scale bar, 50 μm.

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Subcellular localization of ATP synthase revealed by the fractionation method

We previously established the method of subcellular fractionation from Ciona gonad (Ishii & Nishikata 2011). The gonad homogenate is first filtered through the nylon mesh to separate debris and undamaged oocytes. This gonad whole-lysate (W) was centrifuged at low speed (500 g), and stratified into three layers: pellet, supernatant, and fluffy layer, from the centrifugal pole. The pellet and the fluffy layer were designated the insoluble cytoplasmic mass (IC) fraction and fluffy layer (FL) fraction, respectively. The supernatant was then centrifuged at high speed (10 000 g) and the resulting pellet and supernatant were designated the mitochondria enriched (ME) fraction and soluble cytoplasmic (SC) fraction, respectively. The enrichment of mitochondria was always monitored by respiratory activity using the MTT (3-(4,5-dimethyl-thiazole-2-yl)-2,5-diphenyl tetrazolium bromide) assay. We used the fractions with an MTT value in the ME fraction that was at least two times higher than that of the whole lysate, IC and SC fractions.

As shown in Figure 7, the FL fraction contained each of the proteins. We assumed that the FL fraction was a fuzzy, lipid-containing fraction and contaminated with the supernatant of the first centrifugation, which contained ME and SC fractions. aPKC and most of the tubulin was segregated into the SC fraction indicating the effectiveness of this fractionation method to enrich the soluble proteins into the SC fraction. All of the mitochondrial components: ATPα, ATPβ, and ATPγ of the F1-ATP synthase, ATPb of the F0-ATP synthase, PDHE1α, MnSOD, and HSP60 of the matrix component, were highly enriched in the ME fraction. These data also confirmed the effective mitochondrial enrichment of the ME fraction by this method.

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Figure 7. Subcellular localization of each subunit of ATP synthase and mitochondrial marker proteins. Ciona gonad whole lysates (W) were fractionated into four fractions; IC (insoluble cytoplasmic mass), ME (mitochondria enriched), SC (soluble cytoplasmic), and FL (fluffy layer) fractions. In each lane, 2 μg of total protein from each fraction was loaded on a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel, and blotted onto polyvinylidene fluoride (PVDF) membranes. The membranes were probed with anti-ATPα antibody (ATPα), anti-ATPβ antibody (ATPβ), anti-ATPγ antibody (ATPγ), anti-ATPb antibody (ATPb), anti-PDHE1α antibody (PDHE1α), anti-MnSOD antibody (MnSOD), anti-GroEL antibody (HSP60), anti-aPKC antibody (aPKC), and anti-β tubulin antibody (β-tubulin). Arrowhead indicates the bands of the different size, which were observed with the preimmune serum of the anti-ATPb antibody.

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Finally, in the IC fraction, although faint bands were observed for the mitochondrial proteins, considerable amounts of ATPα and ATPβ were detected. As the same amount of protein from each fraction was loaded in each lane, and the total amount of protein in each fraction was measured, the recoveries of these protein bands in each fraction from gonad-whole lysate could be estimated from the band intensity. The recovery of MnSOD from whole-gonad homogenate was 49 ± 8.2% in the ME, but only 14 ± 7.0% in the IC. In the case of ATPα and ATPβ, the recovery in the ME was 33 ± 2.7% and 24 ± 1.6%, while the recovery in the IC was 35 ± 6.2 and 35 ± 11.5%, respectively. Assuming that the recovery of MnSOD reflects mitochondria content of each fraction, the high level of ATPα and ATPβ recovery to the IC fraction indicates that about 20% of the total amount of ATPα and ATPβ is located exomitochondrially.

Interaction between ATP synthase subunits and the cytoskeleton

As p58 was suggested to be a cytoskeletal component (Jeffery & Swalla 1992; Nishikata & Wada 1996), the finding that exomitochondrial ATPα and ATPβ were fractionated into the IC fraction was not surprising. In order to confirm that the exomitochondrial ATPα bound to the cytoskeleton, we carried out the following experiment with the non-ionic detergent Triton X-100. Many studies reported that ascidian eggs treated with Triton X-100 could well retain cytoskeletal components (e.g. Swalla et al. 1991; Nishikata & Wada 1996). On the other hand, many studies, in which the mammalian mitochondrial ATP synthase could be solubilized with some kind of detergents, including Triton X-100, have been made (e.g. Linnett et al. 1975; Soper & Pedersen 1976). In particular, Ko et al. (2003) clearly showed that about 0.5% of Triton X-100 completely solubilizes the entire ATP synthase from rat mitochondrial inner membrane.

We extracted unfertilized eggs with 2% Triton X-100. Figure 8A shows a Western blot of the total protein from whole eggs and detergent-extracted eggs. The same amount of total protein was loaded in each lane. While most of the MnSOD was extracted from the egg, ATPα and ATPβ persisted within the egg. Similar to MnSOD, most of the PDHE1α, HSP60, ATPγ, and ATPb in the unfertilized egg were solubilized with Triton X-100 (data not shown). The same batch of whole and extracted unfertilized eggs was also immunohistochemically stained with anti-ATPα and anti-MnSOD antibodies (Fig. 8B–G). In the extracted egg, the remaining ATPα was stayed in the cortical region of the unfertilized egg. However, MnSOD was hardly detectable in the egg. On the basis of Ko's experiments (2003), ascidian mitochondrial ATPα would be solubilized in our condition. Moreover, the components insolublized in our condition would be cytoskeleton. So, our results suggested that the exomitochondrial ATPα was bound to the egg cytoskeleton.

image

Figure 8. Different detergent solubility of mitochondrial proteins within the egg. The unfertilized eggs were treated with a non-ionic detergent, Triton X-100, as described in materials and methods. (A) The same amount (2 μg) of proteins from intact egg and extracted egg were loaded on an sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)-gel, and blotted onto polyvinylidene fluoride (PVDF) membranes. The membranes were probed with anti-ATPα antibody (ATPα), anti-ATPβ antibody (ATPβ), and anti-MnSOD antibody (MnSOD). Most of the MnSOD protein was extracted from the egg. (B–G) Vertical sections of intact (B–D) and extracted (E–G) eggs were stained with anti-ATPα antibody (B, E; ATPα; Rhodamine, red) and anti-MnSOD antibody (C, F; MnSOD; FITC [fluorescein isothiocyanate], green). Merged images are also shown (D, G; Merge). While most of the MnSOD protein was also extracted from the egg myoplasm, the ATPα protein remained in the myoplasm. Scale bar, 50 μm.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Behavior of mitochondrial proteins

Using comprehensive immunohistochemical staining, we categorized two staining patterns: staining with anti-ATPα and anti-ATPβ antibodies: and staining with anti-MnSOD and anti-PDHE1α antibodies. The former are mitochondrial inner membrane proteins and the latter are mitochondrial matrix proteins. The differences observed in the staining patterns might be due to this intramitochondrial localization pattern. As we could not compare the exact localization pattern of mitochondria and these staining patterns, we cannot say which pattern represents the mitochondrial localization pattern. In general, as MnSOD is an anti-oxidant enzyme and its expression is thought to be regulated according to the cell conditions (e.g. Andreyev et al. 2005; Aiken et al. 2008), its localization pattern may not always represent the mitochondria localization pattern. However, in this study, at least at the crescent stage, TEM clearly distinguished a 2- to 6-μm-thick mitochondria-less cortical region from a mitochondria-rich internal region. The latter was congruent with the immunofluorescent staining with anti-MnSOD antibodies. This mitochondria-less cortical region was pointed out by previous studies to be an ER-rich domain (e.g. Speksnijder et al. 1993; Roegiers et al. 1999). As this region is also known as the site for the postplasmic maternal RNAs localization (Yoshida et al. 1996), Sardet et al. (2007) called this region the cortical endoplasmic reticulum and maternal postplasmic/PEM RNAs domain (cER-mRNA domain). From these points of view, at least at the crescent stage, the distribution of MnSOD represents the mitochondria localization pattern and, hence, the intense staining of this cER-mRNA domain with anti-ATPα and anti-ATPβ antibodies was not due to the mitochondrial ATP synthase, but arose from the exomitochondrial ATP synthase subunits.

In previous studies, anti-ATPα antibody (NN18) was used as a marker for the myoplasm (e.g. Swalla et al. 1991; Chiba et al. 1999), and sometimes it was used as a marker for the mitochondria, which localize to the myoplasm (e.g. Prodon et al. 2006, 2007). These studies did not pay much attention to the intense staining in the cER-mRNA domain. In the current study, double staining with anti-ATPα and anti-MnSOD antibodies first clearly highlighted the cER-mRNA domain as the ATPα-enriched and MnSOD-less cortical region. However, the relationship between exomitochondrial ATP synthase subunits and the cortical ER and postplasmic RNAs have yet to be analyzed.

Analyses by the subcellular fractionation method

The subcellular fractionation method used in this study was reliable. Although the FL fraction was contaminated with the ME and SC fractions, the constituent molecules in the IC, ME, and SC fractions were clearly distinct (Fig. 7). This reliability was confirmed by the monitoring of mitochondrial respiratory activity. Using this reliable method, we found that only ATPα and ATPβ were recovered in both IC and ME fractions. The other mitochondrial components, three matrix proteins, one subunit of F0-ATP synthase, and even one subunit of F1-ATP synthase, were mostly recovered in the ME fraction. This result strongly suggests that only ATPα and ATPβ are localized exomitochondrially.

In order to obtain more convincing evidence for the exomitochondrial localization of these proteins, immunoelectron microscopic observations are necessary. In addition, subcellular fractionation of crescent stage embryos and immunoprecipitation experiments with anti-ATPα and anti-ATPβ antibodies will offer very insightful data. Such immunoprecipitation experiments will lead us closer to identifying the binding partner of these exomitochondrial ATP synthase subunits, which will offer a clue to understanding their function.

Association of exomitochondrial ATP synthase subunits with the cytoskeleton

As p58 is believed to be a cytoskeletal component (Jeffery & Swalla 1992) and to bind to the myoplasmin-C1 (Nishikata & Wada 1996), the exomitochondrial ATP synthase subunits were necessarily thought to associate with the cytoskeleton. In the current study, we showed the difference in solubility in detergent between ATPα and MnSOD in the living unfertilized egg; however, the results could be due to differences in the molecular nature of the inner membrane protein and the matrix protein. Although this possibility can not be excluded, the long research history of the mammalian ATP synthase testifies to the solubility of mitochondrial ATP synthase even in 0.5% Triton X-100-containing buffers (Ko et al. 2003). So, the finding in the current study that any of the ATPα was insoluble in the buffers containing a relatively high concentration (2%) of Triton X-100 was unexpected. Our extraction experiments showed that exomitochondrial ATPα in the ascidian egg was bound to the cytoskeleton. This result also supported the existence of the exomitochondrial ATPα.

Novelty of the ascidian exomitochondrial ATP synthase subunits

Exomitochondrial ATP synthase has been reported in mammalian cells. In human umbilical vein endothelial cells (HUVEC), exomitochondrial ATP synthase was located on the plasma membrane and functioned as an angiostatin receptor (Moser et al. 2001; Veitonmäki et al. 2004). In the human hepatocellular liver carcinoma cell line (HepG2) and normal human liver cells, exomitochondrial ATP synthase was located on the plasma membrane and functioned as an apolipoprotein A-1 (apoA-1) receptor (Martinez et al. 2003; Mangiullo et al. 2008). Also, in some human tumor cells, exomitochondrial ATP synthase was located on the plasma membrane and lowered the surrounding pH (Kenan & Wahl 2005). All of these ATP synthases were the complete form of the enzyme complex and, additionally, their ATP hydrolyzing enzyme activities were experimentally proven (Moser et al. 2001; Mangiullo et al. 2008).

Our results are totally new from the viewpoints of the completeness of the enzyme complex and intracellular localization. In this study, we suggest that only a part of the ATP synthase, subunit α and β, is exomitochondrially localized in the cytosol. Moreover, this localized region is the cortical ER-rich region of the myoplasm, at least at the crescent stage. However, the function of the exomitochondrial ATP synthase subunits in the ascidian embryo is still obscure. The localized region, which was the cER-mRNA domain, suggests its involvement in the versatile functions of the myoplasm such as, muscle development (e.g. Nishida & Sawada 2001), embryonic axis formation (e.g. Negishi et al. 2011) and germ cell specification (e.g. Shirae-Kurabayashi et al. 2011).

In the fields of cell biology, it is well known that identical proteins are found in more than one subcellular compartment. It is called “dual targeting” or “dual distribution” of the protein (e.g. Karniely & Pines 2005; Herrmann 2009). For example, the mature form of fumarase (Stein et al. 1994; Soltys & Gupta 1999) and aconitase (Regev-Rudzki et al. 2005) are distributed between the mitochondria and cytosol. In these two examples, the dual targeting mechanism is achieved by the specific folding properties of the C-terminal region, which distinguishes import into the matrix or release back into the cytosol (Knox et al. 1998). One of the exomitochondrial subunits, ATPα, was immunoprecipitated by the NN18 antibody and the N-terminal amino acid sequence was determined (data not shown). The sequence lacked the tentative mitochondria-targeting signal sequence and was thought to represent a matured form. Therefore, ATPα has a possibility to achieve its dual distribution by the same mechanism as fumarase. In this context, we showed a novel dual-targeting protein within the ascidian egg. The large size of the egg facilitates easy discrimination between mitochondria and cytosol. Moreover, the processes of oogenesis and early development, including the cytoplasmic and cortical rearrangement, have been well described. This model could provide a unique and powerful experimental system to analyze the dual targeting mechanism.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Adult C. intestinalis were provided by Kyoto University and University of Tokyo with support from the National Bio-Resource Project (NBRP) of the MEXT, Japan. We thank Dr A. Kijima and all the members of the Onagawa Field Science Center (Tohoku University, Onagawa Bay, Japan); Dr M. Furusho of the Faculty of Maritime Sciences, Kobe University; Dr T. G. Kusakabe of the Faculty of Science and Engineering, Konan University; and Dr Y. Satou and Ms K. Hirayama of Kyoto University for their kind help in collecting animals. This work was supported in part by a Grant-in-Aid for Core Research Projects (2009–2014) from the Ministry of Education, Culture, Sports, Science and Technology, Japan (TN), and the Sasakawa Scientific Research Grant from the Japan Science Society (HI).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • Aiken, K. J., Bickford, J. S., Kilberg, M. S. & Nick, H. S. 2008. Metabolic regulation of manganese superoxide dismutase expression via essential amino acid deprivation. J. Biol. Chem. 283, 1025210263.
  • Andreyev, A. Y., Kushnareva, Y. E. & Starkov, A. A. 2005. Mitochondrial metabolism of reactive oxygen species. Biochemistry (Mosc) 70, 200214.
  • Berg, W. E. & Humphreys, W. J. 1960. Electron microscopy of four-cell stages of the ascidians Ciona and Styela. Dev. Biol. 2, 4260.
  • Chiba, S., Miki, Y., Ashida, K., Wada, M. R., Tanaka, K. J., Shibata, Y., Nakamori, R. & Nishikata, T. 1999. Interactions between cytoskeletal components during myoplasm rearrangement in ascidian eggs. Dev. Growth Differ. 41, 265272.
  • Conklin, E. G. 1905a. Mosaic development in ascidian eggs. J. Exp. Zool. 2, 145223.
  • Conklin, E. G. 1905b. The organization and cell lineage of the ascidian egg. J. Acad. Nat. Sci. Phila. 13, 1119.
  • Fujiwara, A. & Yasumasu, I. 1997. Does the respiratory rate in sea urchin embryos increase during early development without proliferation of mitochondria? Dev. Growth Differ. 39, 179189.
  • Gualtieri, R. & Sardet, C. 1989. The endoplasmic reticulum network in the ascidian egg: localization and calcium content. Biol. Cell 65, 301304.
  • Herrmann, J. M. 2009. Putting a break on protein translocation: metabolic regulation of mitochondrial protein import: MicroCommentary. Mol. Microbiol. 72, 275278.
  • Hibino, T., Nishikata, T. & Nishida, H. 1998. Centrosome-attracting body: a novel structure closely related to unequal cleavages in the ascidian embryo. Dev. Growth Differ. 40, 8595.
  • Iseto, T. & Nishida, H. 1999. Ultrastructural studies on the centrosome-attracting body: electron-dense matrix and its role in unequal cleavages in ascidian embryos. Dev. Growth Differ. 41, 601609.
  • Ishii, H. & Nishikata, T. 2011. Mitochondrial ATP synthase is ectopically localized to the ascidian egg myoplasm. Proceedings of Annual Meeting of Multidirectional Biological Functions 2, 1718.
  • Jeffery, W. R. & Meier, S. 1983. A yellow crescent cytoskeletal domain in ascidian eggs and its role in early development. Dev. Biol. 96, 125143.
  • Jeffery, W. R. & Swalla, B. J. 1992. Factors necessary for restoring an evolutionary change in an anural ascidian embryo. Dev. Biol. 153, 194205.
  • Karniely, S. & Pines, O. 2005. Single translation–dual destination: mechanisms of dual protein targeting in eukaryotes. EMBO Rep. 6, 420425.
  • Kenan, D. . J. & Wahl, M. L. 2005. Ectopic localization of mitochondrial ATP synthase: a target for anti-angiogenesis intervention? J. Bioenerg. Biomembr. 37, 461465.
  • Knox, C., Sass, E., Neupert, W. & Pines, O. 1998. Import into mitochondria, folding and retrograde movement of fumarase in yeast. J. Biol. Chem. 273, 2558725593.
  • Ko, Y. H., Delannoy, M., Hullihen, J., Chiu, W. & Pedersen, P. L. 2003. Mitochondrial ATP synthasome. Cristae-enriched membranes and a multiwell detergent screening assay yield dispersed single complexes containing the ATP synthase and carriers for Pi and ADP/ATP. J. Biol. Chem. 278, 1230512309.
  • Kumano, G., Takatori, N., Negishi, T., Takada, T. & Nishida, H. 2011. A maternal factor unique to ascidians silences the germline via binding to P-TEFb and RNAP II regulation. Curr. Biol. 21, 13081313.
  • Lambert, C. C. & Epel, D. 1979. Calcium-mediated mitochondrial movement in ascidian sperm during fertilization. Dev. Biol. 69, 296304.
  • Lambert, C. C. & Koch, R. A. 1988. Sperm binding and penetration during ascidian fertilization. Dev. Growth Differ. 30, 325336.
  • Linnett, P. E., Mitchell, A. D. & Beechey, R. B. 1975. Changes in inhibitor sensitivity of the mitochondrial ATPase activity after detergent solubilisation. FEBS Lett. 53, 180183.
  • Makabe, K. W., Kawashima, T., Kawashima, S., Minokawa, T., Adachi, A., Kawamura, H., Ishikawa, H., Yasuda, R., Yamamoto, H., Kondoh, K., Arioka, S., Sasakura, Y., Kobayashi, A., Yagi, K., Shojima, K., Kondoh, Y., Kido, S., Tsujinami, M., Nishimura, N., Takahashi, M., Nakamura, T., Kanehisa, M., Ogasawara, M., Nishikata, T. & Nishida, H. 2001. Large-scale cDNA analysis of the maternal genetic information in the egg of Halocynthia roretzi for a gene expression catalog of ascidian development. Development 128, 25552567.
  • Mangiullo, R., Gnoni, A., Leone, A., Gnoni, G. V., Papa, S. & Zanotti, F. 2008. Structural and functional characterization of F(o)F(1)-ATP synthase on the extracellular surface of rat hepatocytes. Biochim. Biophys. Acta 1777, 13261335.
  • Martinez, L. O., Jacquet, S., Esteve, J. P., Rolland, C., Cabezón, E., Champagne, E., Pineau, T., Georgeaud, V., Walker, J. E., Tercé, F., Collet, X., Perret, B. & Barbaras, R. 2003. Ectopic β-chain of ATP synthase is an apolipoprotein A-I receptor in hepatic HDL endocytosis. Nature 421, 7579.
  • Moser, T. L., Kenan, D. J., Ashley, T. A., Roy, J. A., Goodman, M. D., Misra, U. K., Cheek, D. J. & Pizzo, S. V. 2001. Endothelial cell surface F1-FO ATP synthase is active in ATP synthesis and is inhibited by angiostatin. Proc. Natl. Acad. Sci. U S A 98, 66566661.
  • Negishi, T., Kumano, G. & Nishida, H. 2011. Polo-like kinase 1 is required for localization of Posterior End Mark protein to the centrosome-attracting body and unequal cleavages in ascidian embryos. Dev. Growth Differ. 53, 7687.
  • Negishi, T., Takada, T., Kawai, N. & Nishida, H. 2007. Localized PEM mRNA and protein are involved in cleavage-plane orientation and unequal cell divisions in ascidians. Curr. Biol. 17, 10141025.
  • Nishikata, T., Mita-Miyazawa, I., Deno, T. & Satoh, N. 1987. Monoclonal antibodies against components of the myoplasm of eggs of the ascidian Ciona intestinalis partially block the development of muscle-specific acetylcholinesterase. Development 100, 577586.
  • Nishikata, T. & Wada, M. 1996. Molecular characterization of myoplasmin-C1: a cytoskeletal component localized in the myoplasm of the ascidian egg. Dev. Genes. Evol. 206, 7279.
  • Nishikata, T., Hibino, T. & Nishida, H. 1999. The centrosome-attracting body, microtubule system, and posterior egg cytoplasm are involved in positioning of cleavage planes in the ascidian embryo. Dev. Biol. 209, 7285.
  • Nishida, H. & Sawada, K. 2001. macho-1 encodes a localized mRNA in ascidian eggs that specifies muscle fate during embryogenesis. Nature 409, 724729.
  • Patalano, S., Prulière, G., Prodon, F., Paix, A., Dru, P., Sardet, C. & Chenevert, J. 2006. The aPKC-PAR-6-PAR-3 cell polarity complex localizes to the centrosome attracting body, a macroscopic cortical structure responsible for asymmetric divisions in the early ascidian embryo. J. Cell Sci. 119, 15921603.
  • Prodon, F., Dru, P., Roegiers, F. & Sardet, C. 2005. Polarity of the ascidian egg cortex and relocalization of cER and mRNAs in the early embryo. J. Cell Sci. 118, 23932404.
  • Prodon, F., Chenevert, J. & Sardet, C. 2006. Establishment of animal-vegetal polarity during maturation in ascidian oocytes. Dev. Biol. 290, 297311.
  • Prodon, F., Yamada, L., Shirae-Kurabayashi, M., Nakamura, Y. & Sasakura, Y. 2007. Postplasmic/PEM RNAs: a class of localized maternal mRNAs with multiple roles in cell polarity and development in ascidian embryos. Dev. Dyn. 236, 16981715.
  • Regev-Rudzki, N., Karniely, S., Ben-Haim, N. N. & Pines, O. 2005. Yeast aconitase in two locations and two metabolic pathways: seeing small amounts is believing. Mol. Biol. Cell 16, 41634171.
  • Roegiers, F., Djediat, C., Dumollard, R., Rouvière, C. & Sardet, C. 1999. Phases of cytoplasmic and cortical reorganizations of the ascidian zygote between fertilization and first division. Development 126, 31013117.
  • Sardet, C., Nishida, H., Prodon, F. & Sawada, K. 2003. Maternal mRNAs of PEM and macho 1, the ascidian muscle determinant, associate and move with a rough endoplasmic reticulum network in the egg cortex. Development 130, 58395849.
  • Sardet, C., Dru, P. & Prodon, F. 2005. Maternal determinants and mRNAs in the cortex of ascidian oocytes, zygotes and embryos. Biol. Cell 97, 3549.
  • Sardet, C., Paix, A., Prodon, F., Dru, P. & Chenevert, J. 2007. From oocyte to 16-cell stage: cytoplasmic and cortical reorganizations that pattern the ascidian embryo. Dev. Dyn. 236, 17161731.
  • Satoh, N., Deno, T., Nishida, H., Nishikata, T. & Makabe, K. W. 1990. Cellular and molecular mechanisms of muscle cell differentiation in ascidian embryos. Int. Rev. Cytol. 122, 221258.
  • Satoh, N. 1994. Developmental Biology of Ascidians. Cambridge University Press, Cambrige.
  • Svensson, O. L. 2010. Mitochondria: Structure, Functions and Dysfunctions. Nova Science Publishers, Inc., New York.
  • Shimai, K., Ishii, H. & Nishikata, T. 2010. Interaction between oocyte and accessory cells during ascidian oogenesis. Mem. Konan Univ., Sci. Eng. Ser. 6, 3141.
  • Shirae-Kurabayashi, M., Nishikata, T., Takamura, K., Tanaka, K. J., Nakamoto, C. & Nakamura, A. 2006. Dynamic redistribution of vasa homolog and exclusion of somatic cell determinants during germ cell specification in Ciona intestinalis. Development 133, 26832693.
  • Shirae-Kurabayashi, M., Matsuda, K. & Nakamura, A. 2011. Ci-Pem-1 localizes to the nucleus and represses somatic gene transcription in the germline of Ciona intestinalis embryos. Development 138, 28712881.
  • Soltys, B. J. & Gupta, R. S. 1999. Mitochondrial-matrix proteins at unexpected locations: are they exported? Trends Biochem. Sci. 24, 174177.
  • Soper, J. W. & Pedersen, P. L. 1976. Adenosine triphosphatase of rat liver mitochondria: detergent solubilization of an oligomycin- and dicyclohexylcarbodiimide-sensitive form of the enzyme. Biochemistry 15, 26822690.
  • Steedman, H. F. 1957. Polyester wax; a new ribboning embedding medium for histology. Nature 179, 1345.
  • Stein, I., Peleg, Y., Even-Ram, S. & Pines, O. 1994. The single translation product of the FUM1 gene (fumarase) is processed in mitochondria before being distributed between the cytosol and mitochondria in Saccharomyces cerevisiae. Mol. Cell. Biol. 14, 47704778.
  • Speksnijder, J. E., Terasaki, M., Hage, W. J., Jaffe, L. F. & Sardet, C. 1993. Polarity and reorganization of the endoplasmic reticulum during fertilization and ooplasmic segregation in the ascidian egg. J. Cell Biol. 120, 13371346.
  • Spurr, A. R. 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 3143.
  • Swalla, B. J.,  Badgett, M. R. & Jeffery, W. R. 1991. Identification of a cytoskeleton protein localized in the myoplasm of ascidian eggs: localization is modifed during anural development. Development 111, 425436.
  • Veitonmäki, N., Cao, R., Wu, L. H., Moser, T. L., Li, B., Pizzo, S. V., Zhivotovsky, B. & Cao, Y. 2004. Endothelial cell surface ATP synthase-triggered caspase-apoptotic pathway is essential for K1-5-induced antiangiogenesis. Cancer Res. 64, 36793686.
  • Yoshida, S., Marikawa, Y. & Satoh, N. 1996. Posterior end mark, a novel maternal gene encoding a localized factor in the ascidian embryo. Development 122, 20052012.