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Effects of Fcj1-Mos1 and mitochondrial division on aggregation of mitochondrial DNA nucleoids and organelle morphology

Mitochondrial DNA nucleoids are distributed as many discrete foci in mitochondria. Nucleoid distribution is controlled by mitochondrial division and Fcj1 and Mos1, two evolutionarily conserved, mitochondrial proteins that maintain cristae junctions and tubular organelle morphology.


INTRODUCTION
Mitochondria possess their own genome, called mitochondrial DNA (mtDNA), which encodes several essential components of oxidative phosphorylation. mtDNA is exposed to oxidative stress in mitochondria, and its mutations are associated with many human diseases (Wallace, 2010;Nunnari and Suomalainen, 2012). Like chromosomes for the nuclear genome, the mitochondrial genome is packaged into nucleoprotein complexes called nucleoids to protect from deleterious, oxidative damage (Chen and Butow, 2005;Spelbrink, 2010). Nucleoids are also important for the biogenesis of mtDNA, as they contain proteins that mediate DNA replication, repair, and recombination. Proteomic studies have identified >50 different proteins associated with nucleoids (Kaufman et al., 2000;Wang and Bogenhagen, 2006). Defects in these nucleoid proteins result in loss of mtDNA, demonstrating the importance of nucleoids for the maintenance of mtDNA (Gilkerson, 2009;Solieri, 2010).
Cells have multiple copies of mtDNA, and its copy number varies depending on the cell type. For example, yeast haploid cells contain ∼30 copies, whereas human cells have ∼300 copies (Chen and Butow, 2005;Spelbrink, 2010). mtDNA nucleoids are relatively uniform in shape and size. In yeast cells, one or two copies of mtDNA are packaged into each nucleoid (MacAlpine et al., 2000). Nucleoids are visualized as small discrete foci of ∼0.3 μm in diameter along mitochondrial tubules when stained by DNA-binding fluorescent dyes (MacAlpine et al., 2000). Such distribution of nucleoids ensures that each mitochondrion carries mtDNA (Chen and Butow, 2005). In addition, even distribution of nucleoids also facilitates efficient transmission of mtDNA into daughter cells during cell division. Because mtDNA cannot be synthesized de novo, faithful inheritance of mtDNA is essential for the maintenance of mitochondrial functions. Fcj1 and Mos1 are required for maintaining the size of mtDNA nucleoids. Although Fcj1 also interacts with Mia40, an intermembrane space (IMS) protein that mediates protein import (von der Malsburg et al., 2011), the depletion of Mia40 did not change nucleoid size (Supplemental Figure S2), showing that the nucleoid phenotype is not due to import defects in fcj1∆ cells.
When we generated fcj1∆mos1∆ double-deletion cells, the number of cells with larger (>0.5 μm) mtDNA nucleoids increased to ∼12%, with 40% of them exhibiting a single large mtDNA nucleoid with diameter of >1.0 μm (Figure 1, A and B). These results suggest partially overlapping functions for Fcj1 and Mos1 in the maintenance of mtDNA nucleoid size. In fcj1∆, mos1∆, and fcj1∆mos1∆ cells, mtDNA was still associated with Abf2, an HMG box-containing, DNA-binding protein required for packaging mtDNA into nucleoids (Miyakawa et al., 2010), as DAPI staining colocalized with Abf2-green fluorescent protein (GFP; Figure 1D). Therefore the enlarged morphology of mtDNA nucleoids was not due simply to dissociation of Abf2 from mtDNA. Finally, Southern blotting showed that WT, fcj1∆, mos1∆, and fcj1∆mos1∆ cells contained similar amounts of mtDNA (Supplemental Figure S3). Thus increased size of nucleoids appears not to affect the maintenance of mtDNA in these mutants.

Fcj1 is located adjacent to mtDNA nucleoids
To examine the spatial relationship of mtDNA nucleoids with Fcj1 and Mos1, we replaced chromosomal FCJ1 and MOS1 by FCJ1-GFP and MOS1-GFP, respectively. GFP was fused to Fcj1 and Mos1 at their C-terminus, which faces the IMS. Both Fcj1-GFP and Mos1-GFP were functional, as cells expressing these GFP fusions showed normal morphology of mitochondria and nucleoids ( Figure 2A) and grew normally (Supplemental Figure S4). A fraction of Fcj1-GFP formed punctate structures in mitochondria, with 80% located adjacent to mtDNA nucleoids (n = 120; Figure 2A). The formation of Fcj1-GFP puncta depended on Mos1, but not Aim5, Aim13, Aim37, or Mos2 ( Figure 2B). In contrast, Mos1-GFP was uniformly distributed in mitochondria, similar to an IMS protein, Ups1-GFP ( Figure  2A; Sesaki et al., 2006;Tamura et al., 2009). Because FCJ1 and MOS1 deletion has additional effects on nucleoid size, Mos1 likely has roles in addition to Fcj1-GFP puncta formation. Consistent with our finding that Fcj1-GFP puncta is closely positioned to nucleoids, a proteomic study showed that Fcj1 binds to the nucleoid protein Abf2 in yeast (von der Malsburg et al., 2011). Another study showed that mtDNA nucleoids purified from HeLa cells contained a mammalian homologue of Fcj1, mitofilin (Wang and Bogenhagen, 2006). However, we found that Fcj1 and Mos1 do not associate with nucleoids in sucrose density gradient centrifugation and that Abf2 does not interact with Fcj1 or Mos1 in coimmunoprecipitation studies (unpublished observations). Therefore either only a small fraction of Fcj1 or Mos1 bind to nucleoids or their interactions might be labile.
Mitochondria form large, hollow spheres in fcj1∆ and mos1∆ cells that contain large mtDNA nucleoids When we examined mitochondrial morphology in fcj1∆, mos1∆, and fcj1∆mos1∆ cells that contain large mtDNA nucleoids (>1.0 μm in diameter), essentially all of the cells displayed previously uncharacterized, enlarged spherical mitochondria (Figure 3, A and B). These mitochondria appeared to be hollow by matrix-targeted Su9red fluorescent protein (RFP) and had mtDNA nucleoids around the rim. The spherical structures were clearly observed by differential interference contrast (DIC) microscopy ( Figure 3A, asterisks). To further characterize the hollow, spherical mitochondria, we targeted Despite its importance, the molecular mechanisms that control the distribution of mtDNA nucleoids are largely unknown. Here we investigate how the size and distribution of mtDNA nucleoids are maintained.

Fcj1 and Mos1 are required for maintaining mtDNA nucleoid size
To identify mitochondrial components that control the size and distribution of mtDNA nucleoids, we selected 69 strains with reported alterations in mtDNA maintenance and/or inheritance from a yeast collection in which individual nonessential genes are deleted. These deletion strains include aim∆ (altered inheritance of mitochondria), rrg∆ (required for respiratory growth), and fmp∆ mutants (found in mitochondrial proteome; Supplemental Table S1; Sickmann et al., 2003;Hess et al., 2009;Merz and Westermann, 2009). To visualize mtDNA nucleoids, we incubated live yeast cells with 4′,6-diamidino-2-phenylindole (DAPI), which preferentially labels mtDNA in the absence of chemical fixation (Adams et al., 1997). Most strains (i.e., 63 strains) displayed normal-sized mtDNA nucleoids. However, 5 strains (aim10∆, aim15∆, rrg2∆, rrg4∆, and rrg8∆) exhibited decreased amounts of mtDNA, but their nucleoid size was unaffected (unpublished observations). More important, a strain lacking the FCJ1 gene (also called AIM28) possessed larger nucleoids. We generated independent fcj1∆ strains and examined their phenotypes. In wild-type (WT) cells, the average diameter of mtDNA nucleoids was ∼0.3 μm, with structures >0.5 μm not observed. However, 7% of fcj1∆ cells displayed nucleoids >0.5 μm in diameter ( Figure 1, A and B). Among them, ∼15% of fcj1∆ cells contained a single or a few large mtDNA nucleoids with diameter of >1.0 μm. Total fluorescence intensity was greater in these large mtDNA nucleoids, suggesting that these larger structures may result from nucleoid aggregation or incomplete nucleoid division ( Figure 1A). Overexpression of Fcj1 from the GAL1 promoter did not affect nucleoid size (Supplemental Figure S1). mtDNA is located near the mitochondriaendoplasmic reticulum (ER) contact sites between these organelles and observed next to the tethering complex containing Mmm1, Mmm2, Mdm10, and Mdm12 (Youngman et al., 2004;Kornmann et al., 2009;Nguyen et al., 2012). In cells lacking these proteins, mtDNA do not form nucleoids and diffuse in the matrix. We found that enlarged nucleoids are still located next to Mmm1-GFP in fcj1∆ cells ( Figure 1C), suggesting that the mitochondria-ER contact site remained in fcj1∆ cells and enlarged nucleoids do not result from the loss of the contact site.
To understand whether the formation of hollow, spherical mitochondria requires mtDNA, we examined mitochondrial morphology in fcj1∆mos1∆ cells with (rho + ) and without (rho 0 ) mtDNA. As described, ∼5% of rho + fcj1∆mos1∆ cells possessed enlarged, spherical mitochondria (Figure 3, E and F). However, these large, spherical mitochondria were missing in rho 0 fcj1∆mos1∆ cells. Moreover, lamellar structures of mitochondria were not found in rho 0 fcj1∆mos1∆ cells, consistent with previous observations showing that dimerization of F1Fo-ATP synthase mediated by Atp20 and Atp21 (therefore mtDNA that encodes subunits of this enzyme complex) is required for the formation of lamellar mitochondria (Hoppins et al., 2011). These results suggest that the generation of enlarged, spherical mitochondria requires mtDNA and that hollow, spherical mitochondria might derive from lamellar mitochondria.

Loss of Atp20 and Atp21 rescues nucleoid defects in cells lacking Fcj1 and Mos1
Because Atp20 and Atp21 control the structure of cristae membranes and loss of these proteins suppresses growth defects in fcj1∆ cells (Rabl et al., 2009), we examined nucleoid morphology in cells lacking these proteins. atp20∆ and atp21∆ cells exhibited normalsized nucleoids (Figure 4, A and B). It is striking, however, that loss of Atp20 or Atp21 rescued nucleoid defects in fcj1∆mos1∆ cells (Figure 4, A and B). The inhibitor of F1Fo-ATP synthase, oligomycin, did not affect nucleoid size in fcj1∆mos1∆ cells, showing that dimerization of this enzyme, rather than its activity, is important (Supplemental Figure S5). In terms of mitochondrial morphology, atp20∆ and atp21∆ cells displayed lamellar or partially fragmented mitochondria ( Figure 4C). As described earlier, fcj1∆mos1∆ mitochondria displayed lamellar sheets, hollow spheres, and small fragments ( Figures 1A and 3A). These morphological defects were mutually suppressed in fcj1∆mos1∆atp20∆ and fcj1∆mos1∆atp21∆ cells (Figure 4, A and C). Taken together, these results suggest that Fcj1-Mos1 and Atp20-Atp21 play opposite roles in determining nucleoid size and organelle shape.
Blocking mitochondrial division exacerbates nucleoid defects in fcj1∆, mos1∆, and fcj1∆mos1∆ cells Large nucleoids might arise from incomplete division or aggregation of nucleoids in cells lacking Fcj1 and Mos1. To distinguish these two possibilities, we examined nucleoids in cells defective in mitochondrial division. We reasoned that, if nucleoid division decreased, connecting mitochondria would not affect nucleoid size. In contrast, if nucleoids became aggregated, connecting mitochondria might accelerate nucleoid aggregation. We disrupted the DNM1 gene, which is required for mitochondrial division, in fcj1∆, mos1∆, and fcj1∆mos1∆ cells. dnm1∆ cells contained a single mitochondrion with interconnected tubules due to an imbalance that favors fusion over division and normal size of nucleoids, as reported (Bleazard et al., 1999;Sesaki and Jensen, 1999;Figure 5, A and B). We found that additional loss of Dnm1 increased the number of larger nucleoids to 35-40% in dnm1∆fcj1∆, dnm1∆mos1∆, and dnm1∆fcj1∆mos1∆ cells ( Figure 5B), compared with 7-12% in fcj1∆, mos1∆, and fcj1∆mos1∆ cells ( Figure 1B). In contrast, only slight increases in the number of enlarged nucleoids were observed in dnm1∆aim5∆, dnm1∆aim13∆, dnm1∆aim37∆, and dnm1∆mos2∆ some overlap with DAPI staining, similar to the matrix-targeted Su9-RFP. Remarkably, none of these markers labeled the interior of the spherical mitochondria. As expected, markers for vacuoles (FM4-64; Molecular Probes, Eugene, OR), the ER (Sec63-GFP), and the cytosol (GFP) did not label these spherical structures in fcj1∆mos1∆ cells ( Figure 3D). This hollow mitochondrial sphere is, to the best of our knowledge, a novel morphology not reported in any yeast mutants examined previously.
The frequency of spherical mitochondria increased in fcj1∆mos1∆ cells (∼5%) compared with fcj1∆ and mos1∆ cells (∼1%; Figure 3B), for the separation of individual nucleoids, and loss of these proteins might induce nucleoid aggregation. It appears that more nucleoids were gathered and formed large nucleoids when mitochondrial cells (Supplemental Figure S6A). We also observed synergistic increases in diameter of nucleoids in cells lacking Fcj1, Mos1, and Dnm1 ( Figure 5C). Thus Fcj1 and Mos1 are most likely important  Figure S7). Lamellar mitochondria were still observed in the tripleknockout cells, although it was technically difficult to clearly distinguish net-like mitochondria from the sheet-like mitochondria.

DISCUSSION
Fcj1 and Mos1 are involved in the maintenance of crista junctions, which may be critical for mtDNA nucleoid distribution. For example, in WT mitochondria, organized, periodic arrays of cristae membranes along the boundary membrane may function as partitions to make small compartments for mtDNA nucleoids in the matrix. When these partitions are greatly decreased in cells lacking Fcj1 and Mos1, a small but significant fraction of mtDNA nucleoids aggregate and increase in size. mos2∆, aim5∆, aim13∆, and aim37∆ cells modestly decrease the number of cristae junctions and maintain normal nucleoids. Therefore there may be a threshold for the density of crista junctions required for the maintenance of nucleoid distribution. Second, it is also possible that tubular morphology of mitochondria helps keep nucleoids from aggregation, since Fcj1 and Mos1 also function as tubule-forming components in mitochondria. In sheet mitochondria, nucleoids may collide more frequently due to increases in the direction of their movements in the matrix. Third, in fcj1∆ and mos1∆ cells, cristae membranes that are detached from the boundary membrane may push nucleoids toward the edge of lamellar sheets, leading to aggregation of nucleoids. Finally, nucleoids may be anchored to the IM to keep nucleoids in such small compartments formed by crista membranes, since mtDNA nucleoids are located adjacent to Fcj1. Whether Fcj1 and Mos1 mediate this potential anchoring mechanism awaits further studies. In the absence of division is blocked in cells lacking Fcj1 and/or Mos1. Because a significant fraction of fcj1∆mos1∆dnm1∆ cells still has normal-sized nucleoids, there are likely additional mechanisms by which mtDNA nucleoids remain separate from each other to maintain their distribution within mitochondria.
As observed for fcj1∆ and mos1∆ cells, increased nucleoid size in fcj1∆dnm1∆ cells was decreased with the additional loss of Atp20 or Atp21 ( Figure 5, A and F). Moreover, the formation of hollow, spheri-  is consistent with previous observations showing that nucleoids are dynamic and mobile in mammalian mitochondria (Garrido et al., 2003;Legros et al., 2004). Given that we found that cells Fcj1 and Mos1, mitochondrial division appears to help the separation of mtDNA nucleoids by creating physically separated organelles, maintaining nucleoids in separate mitochondria. This model The nucleoid phenotypes of cells lacking Fcj1 and Mos1 appear to be different from those observed in several mutants with previously reported increased mtDNA nucleoid size. In yeast, Hsp60, a mitochondrial heatshock protein associated with nucleoids, was proposed to participate in nucleoid division (Kaufman et al., 2003). In temperature-sensitive mutants of Hsp60, nucleoids are larger and exhibit a dumbbell-shaped morphology reminiscent of incomplete nucleoid division. In addition, cells lacking Abf2 showed defects in nucleoid packaging and therefore contained swollen nucleoids (Miyakawa et al., 2010). Mmm1, Mmm2, Mdm10, and Mdm12, which have been suggested to participate in ER-mitochondria tethering, mitochondrial shape, and phospholipid metabolism, also regulate mtDNA nucleoids (Kornmann et al., 2009;Nguyen et al., 2012). However, cells lacking Mmm1, Mmm2, Mdm10, and Mdm12 display diffusely distributed mtDNA and Abf2 within the matrix of their large mitochondria, suggesting that these four proteins may also be involved in the packaging of mtDNA into nucleoids (Youngman et al., 2004).
How the hollow mitochondrial sphere is formed in the absence of Fcj1 and Mos1 remains to be determined. We speculate that cristae membranes disconnected from the boundary membrane may fuse with each other and create additional compartments inside mitochondria. This additional compartment is not accessible by proteins imported from the cytosol, as indicated by the lack of staining on the interior of these spheres by the markers we tested. Note that the large, round mitochondria in fcj1∆ and mos1∆ cells are different from those found in mmm1∆, mmm2∆, mdm10∆, mdm12∆, and mdm33∆ cells, as the interiors could be stained with matrix markers (Berger et al., 1997;Hobbs et al., 2001;Messerschmitt et al., 2003). In addition, the formation of large, spherical mitochondria depends on the presence of mtDNA in fcj1∆ and mos1∆ cells, whereas mitochondrial shape in mmm1∆, mmm2∆, mdm10∆, and mdm12∆ cells is independent of mtDNA.

Strains and media
Yeast strains, plasmids, and primers used in this study are listed in Supplemental Table  S2. Disruption of complete open reading frame was performed using the HIS3, kanMX4, and URA3 genes as disruption markers as described (Sikorski and Hieter, 1989;Brachmann et al., 1998). Cells that carry kanMX4 were selected on YPD (1% yeast extract, 2% polypeptone, and 2% glucose) containing 500 μg/ml G418. rho 0 cells were generated by incubating in YPD medium with 25 μg/ml ethidium bromide only partially lose their ability to maintain normal mtDNA nucleoid size in the absence of Fcj1, Mos1, and Dnm1, there are likely additional mechanisms that control the size of mtDNA nucleoids that function alongside these proteins. It will be important to identify these mechanisms in future studies.  for 2 d. Cells were grown in YPD medium, SD medium (0.67% yeast nitrogen base without amino acids and 2% glucose), SCD medium (0.67% yeast nitrogen base without amino acids, 0.5% casamino acid, and 2% glucose), and SGalSuc medium (0.67% yeast nitrogen base without amino acids, 2% galactose, and 2% sucrose) with appropriate supplements (Adams et al., 1997).

Microscopy
Cells were viewed on an Olympus BX61 upright microscope (Olympus, Tokyo, Japan). Fluorescence and DIC images were captured with a CoolSnap HQ charge-coupled device (CCD) camera (Roper Photometrics, Tucson, AZ) using SlideBook software, version 5.0 (Intelligent Imaging Innovations, Denver, CO) and processed with Photoshop CS3 (Adobe, San Jose, CA; Tamura et al., 2010). For electron microscopy, cells were fixed, processed, and embedded in Epon resin, as described (Bauer et al., 2001). Ultrathin sections were obtained using an Ultracut E (Reichert-Jung, Germany), stained with 2% uranyl acetate and lead citrate, and viewed on a Hitachi H-7600 transmission electron microscope (Hitachi, Tokyo, Japan) equipped with the dual AMT CCD camera (Wakabayashi et al., 2009;Kageyama et al., 2012).

Southern blotting
DNA was extracted from yeast cells using the Yeast DNA Extraction Kit (Thermo Scientific, Waltham, MA). Then the DNA (5 μg) was digested with EcoRI, resolved by electrophoresis, and transferred to nitrocellulose membrane. Hybridization was performed using the ECL Direct Nucleic Acid Labelling and Detection System (Amersham, Piscataway, NJ) according to the manufacturer's instructions. The probe used for mtDNA corresponds to position 245-676 of the ATP6 gene, and the PCR primers corresponding to this probe were 5′-TGCTTAAAGGACAAATTGGAGG-3′ and 5′-TCATAGCTAAAGG-TACAAAACCGA-3′. The probe used for nuclear DNA corresponds to position 399-779 of the 18S rRNA gene (RDN18), and the primers corresponding to this probe were 5′-AAACGGCTACCACATC-CAAG-3′ and 5'-ACGCCTGCTTTGAACACTCT-3′.