Mitochondria generate most of the energy used by cells, and they also play key roles in cellular growth, death, and differentiation. They are evolutionarily derived from bacteria and have retained their own DNA and protein translation system, but they are also dependent on the cell for their growth and replication.

A significant portion of the outer membrane of a mitochondrion is in contact with the endoplasmic reticulum (ER)—an organelle that is the starting point for the synthesis of secreted proteins, and is also critical for the synthesis of lipids and other organelles. Recent work suggests that mitochondria–ER contact points mark sites of mitochondrial division, but it is unclear exactly how this process occurs.

Here, Murley et al. use the budding yeast and model organism Saccharomyces cerevisiae to show that at mitochondrial division sites, a multiprotein complex called ERMES promotes the formation of ER–mitochondrial contact points, while an evolutionarily conserved enzyme, Gem1, antagonizes these contacts to aid mitochondrial segregation. The contact points are found adjacent to nucleoids (which are complexes of mitochondrial DNA and proteins)—an observation suggesting that ER-associated mitochondrial division evolved to help distribute nucleoids between newly formed mitochondria.

The present study also reveals a novel role for the conserved protein Gem1 and could lead researchers to reinvestigate the functions of Miro1/2—the equivalent of Gem1 in higher eukaryotes. Miro1/2 is thought to connect mitochondria to motor proteins, which transports them through the cell along microtubules. Dysfunction of Miro1/2 reduces the mobility of mitochondria, and the work of Murley et al. suggests that this could be a consequence of enhanced contacts between mitochondria and the ER.

The distribution of mitochondria and mitochondrial DNA (mtDNA) is accomplished through the engagement of multiple pathways, including mitochondrial division, fusion, motility, and tethering. Mitochondrial division is mediated by a dynamin-related protein, Dnm1 (in yeast)/Drp1 (in mammals) (Lackner and Nunnari, 2009), which self-assembles in a regulated manner on the surface of the mitochondrial outer membrane into helices that mediate mitochondrial scission (Ingerman et al., 2005; Mears et al., 2011). Dnm1 and Drp1 form helices in vitro, whose diameters are significantly smaller than the diameter of unconstricted mitochondria (Ingerman et al., 2005; Bossy et al., 2010). This observation suggests that a mechanism for Dnm1/Drp1-independent constriction may exist to facilitate helix assembly as a first step toward mitochondrial division.

We recently discovered that ER tubules wrap around mitochondria and mark a majority of mitochondrial division sites in yeast and mammalian cells (Friedman et al., 2011). These findings suggest that the process of ‘ER-associated Mitochondrial Division’ (ERMD) facilitates the creation of mitochondrial constriction sites, or geometric ‘hot spots’, for Dnm1/Drp1 helix assembly. Consistent with this model, the association of ER tubules with mitochondrial constriction sites is independent of mitochondrial division components (Friedman et al., 2011). ERMD is also independent of known ER tubule-shaping proteins and Mfn2-mediated ER–mitochondria contacts (de Brito and Scorrano, 2008; Friedman et al., 2011). Indeed, the composition and biogenesis of ERMD sites are unknown.

A candidate for tethering ER and mitochondria at ERMD sites is the ER-Mitochondria Encounter Structure (ERMES)—a multiprotein complex localized at an interface between the ER and mitochondria in budding yeast cells (Kornmann et al., 2009; Toulmay and Prinz, 2012). The ERMES complex is composed of four core subunits, each of which is required for the formation of multiple ERMES foci per cell: Mdm10 and Mdm34 are integral to the mitochondrial outer membrane; Mdm12 is predicted to be cytosolic; and Mmm1 is an ER transmembrane protein (Kornmann et al., 2009; Stroud et al., 2011). ERMES is thought to function as a physical ER–mitochondria tether that serves to distribute mitochondria (Boldogh et al., 2003) and to facilitate the exchange of lipids between the two organelles (Kornmann et al., 2009; Voss et al., 2012). ERMES foci are localized adjacent to a subset of nucleoids, which are engaged in replicating DNA (Hobbs et al., 2001; Hanekamp et al., 2002; Meeusen and Nunnari, 2003), suggesting that ERMES plays an active role in nucleoid regulation. In agreement with this notion, deletion of core ERMES components disrupts nucleoid structure and transforms the tubular mitochondrial network into spherical mitochondria with relatively large diameters (Burgess et al., 1994; Youngman et al., 2004). The ERMES-deficient mitochondrial morphology phenotype is epistatic to the characteristic net-like mitochondrial structures that result from the deletion of genes encoding mitochondrial division components (Youngman et al., 2004), consistent with ERMES functioning upstream of the mitochondrial division machinery in mitochondrial distribution.

Cytological, biochemical, and genetic data indicate that the highly conserved Miro GTPase Gem1 is associated with ERMES at steady state (Kornmann et al., 2011; Stroud et al., 2011). In yeast cells lacking Gem1, mitochondria form a distinct spectrum of abnormal structures, from tubules to clustered spheres, mitochondrial distribution into daughter cells is less efficient than that of wild-type mitochondria, and cells lose mitochondrial DNA at a significantly higher frequency (Frederick et al., 2004; Koshiba et al., 2011). Gem1, however, is not an essential structural component of ERMES, as ERMES foci are observed in gem1Δ cells. However, in gem1Δ cells, it has been reported that ERMES foci are larger in size and fewer in number (Kornmann et al., 2011), suggesting that Gem1 acts as a regulator of ERMES.

Here, we show that ERMES and Gem1 are spatially and functionally linked to ERMD. Our data suggest a model where ERMES and Gem1 function in ERMD to facilitate the engagement and resolution of ER–mitochondria contacts, respectively. Our data indicate that Gem1 acts as a negative regulator of ER–mitochondria contacts at ERMD sites and is required for the spatial resolution of newly formed mitochondrial tips generated by a division event. We also provide evidence that ERMD positions division sites adjacent to mitochondrial nucleoids to bias their distribution into newly generated tips following division. Thus, we postulate that ERMD evolved to link the distribution of nucleoids and mitochondria.

To assess whether ERMES foci are spatially linked to mitochondrial division, we simultaneously imaged fluorescent protein (FP)–tagged ERMES components and mitochondria over time in living yeast cells. To this end, we expressed functional C-terminal GFP fusions of Mdm12, Mdm34, or Mmm1 from their respective endogenous loci and labeled mitochondria using mitochondrial matrix-targeted DsRed (mito-DsRed). We observed a majority of mitochondrial division events associated with Mdm12, Mdm34, or Mmm1-labeled ERMES foci, which subsequent to the division segregate to only one of the two newly generated tips in the mitochondrial network (Figure 1A,B). The fraction of division events associated with ERMES foci (54–60%) is significantly higher than that predicted for a random association (approximately 10%), based on the surface area of mitochondria associated with ERMES (Figure 1B and ‘Materials and methods’). The detection of ERMES foci by fluorescence imaging may underestimate ERMES foci associated with mitochondrial division as we observed a positive correlation between the percentage of mitochondrial divisions associated with a given ERMES subunit and its estimated whole-cell abundance (Huh et al., 2003). However, we cannot rule out the possibility that non-ERMES–associated mitochondrial division sites exist in cells.

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In light of a previous report suggesting that the ERMES components Mdm34 and Mmm1 are not strictly colocalized (Youngman et al., 2004), we also addressed whether different types of ERMES complexes/foci exist that could be preferentially associated with mitochondrial division events. Examination of differentially FP-tagged Mdm34 and Mmm1 or Mdm12 indicated that all components colocalize at mitochondrial division sites (Figure 1—figure supplement 1), consistent with the finding that each core ERMES subunit is necessary for assembly of the complex (Kornmann et al., 2009). In addition, these two components associated with the same mitochondrial tip generated by division, indicating that the complex remains intact during the division process (Figure 1—figure supplement 1). Together, these observations demonstrate that mitochondrial division events are spatially linked to the ERMES complex.

We assessed the relationship of ERMES to ERMD by imaging cells expressing Mdm34-yEGFP, an ER marker (DsRed-HDEL), and a mitochondrial matrix-targeted blue fluorescent protein (mito-TagBFP). As shown in a representative series of time lapse images in Figure 1C, we observed ERMES foci at interfaces between the ER and mitochondria and subsequently at ER-associated mitochondrial constriction and eventual division events. Consistent with this observation, the yeast mitochondrial division machine, marked by Dnm1-mCherry, was observed adjacent to Mdm34-labeled ERMES foci at mitochondrial division sites (Figure 1D). Given that ERMES null mutations are epistatic to DNM1 null mutations (Youngman et al., 2004), these observations suggest that ERMES functions early in ERMD at a step distinct from Dnm1 by bridging interactions between the ER and mitochondria.

The conserved Miro GTPase Gem1 associates with the ERMES complex, potentially as a regulatory subunit (Kornmann et al., 2011; Stroud et al., 2011). Consistent with this, a functional GFP-2xFLAG-Gem1 fusion protein expressed in gem1Δ cells localized to mitochondrial-associated foci labeled by the ERMES subunit Mdm34-mCherry (Figure 1E). Similar to ERMES, Gem1-labeled foci were spatially linked to mitochondrial division sites and segregated to only one tip following division (Figure 1E).

To gain insight into the function of Gem1 in ERMD, we characterized the behavior of ERMES and mitochondria in gem1Δ cells. As previously published, we observed that mitochondrial structure in gem1Δ cells is both aberrant and diverse, ranging from tubules to clustered spheres, with both having an increased mitochondrial diameter (Figure 2A) (Frederick et al., 2004). In addition, regardless of the mitochondrial structure type, we observed that ERMES foci in gem1Δ cells were associated with apparent mitochondrial constriction sites, defined as a narrowing and/or resolved separation of the mitochondrial matrix labeled by mito-DsRed (Figure 2A, arrow heads). A vast majority of ERMES-marked mitochondrial constriction sites in gem1Δ cells were stable, typically persisting for the duration of image capture (3–3.5 min) (Figure 2B and Figure 2—figure supplement 1B,C). This behavior was in contrast to wild-type cells, where ERMES-marked mitochondrial constriction sites resolved into two spatially separated tips within 15–60 s (Figure 1A and Figure 2—figure supplement 1A,C). These observations indicate that Gem1 function is required for the resolution of mitochondrial constriction sites associated with division and/or in the subsequent segregation of mitochondrial tips generated by division events.

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We addressed what structural features are important for Gem1’s role in the resolution of ERMES-associated mitochondrial constriction sites in cells. Under our conditions, expression of Gem1 mutants harboring abrogating mutations in either EF-hand I or II motifs or both rescued all mitochondrial phenotypes in gem1Δ cells to a similar extent as wild-type GEM1 (Figure 2—figure supplement 2). This observation is consistent with the published work demonstrating that the EF-hand regions play a relatively minor role in the maintenance of mitochondrial morphology and distribution, with only EF-hand I acting to stabilize Gem1 expression levels (Frederick et al., 2004; Koshiba et al., 2011). In contrast, expression of Gem1S19N or Gem1S462N, whose mutations abrogate the GTPase activity of GTPase domains 1 and 2, respectively, failed to fully rescue mitochondrial morphological phenotypes in gem1Δ cells. The Gem1S19N mutation was the least functional, consistent with previously published observations (Frederick et al., 2004). Abrogating mutations in the first GTPase domain, such as the Gem1S19N mutation, have been shown to reduce the steady-state localization of Gem1 to ERMES foci (Kornmann et al., 2011). Thus, the severe phenotypes associated with Gem1S19N underscores the functional importance of Gem1’s localization to ERMES foci. Significantly, we also observed stable ERMES-associated mitochondrial constrictions in gem1Δ cells expressing either Gem1S19N or Gem1S462N (Figure 2—figure supplements 2D and 3A,B). Thus, our structure function analysis of Gem1 indicates that similar features, specifically both Gem1 GTPase domains, are required for Gem1’s roles in both the maintenance of mitochondrial morphology and in the resolution of ERMES-marked mitochondrial constriction sites. Thus, the resolution of ERMES-associated mitochondrial constriction sites into segregated mitochondrial tips is a central function of Gem1.

We addressed whether the stability of ERMES-marked mitochondrial constriction sites is a consequence of defective mitochondrial division in gem1Δ cells by examining the behavior of the division machine, labeled with Dnm1-mCherry, over time. We observed that stable mitochondrial constriction sites in gem1Δ cells (mito-TagBFP) were persistently associated with ER tubules (GFP-HDEL) (Figure 2C). In addition, Dnm1 puncta localized to these ER-associated mitochondrial constriction sites in a dynamic manner similar to that observed in wild-type cells, associating and dissociating on a 10-s time scale (Figure 2C) (Frederick et al., 2004). These observations indicate that neither ER engagement with nor Dnm1 recruitment to mitochondrial constriction sites is significantly altered in gem1Δ cells, suggesting that mitochondrial division per se is not defective. This conclusion is supported by the observation that in gem1Δ cells lacking Fzo1, an outer mitochondrial membrane dynamin-related protein required for mitochondrial fusion, mitochondria are fragmented, which is a morphology that requires mitochondrial division (Frederick et al., 2004). Thus, taken together, these observations indicate that the stable ERMES-associated mitochondrial constriction sites in gem1Δ cells is a consequence of defective resolution of mitochondrial tips, perhaps as a consequence of altered ER–mitochondria contacts.

To explore this idea, we analyzed wild-type and gem1Δ cells using three-dimensional electron tomography to determine whether mitochondrial constriction sites in gem1Δ cells represent division intermediates or fully resolved division events and to examine ER–mitochondria contact sites at higher resolution. Tomographic analysis revealed that, in contrast to tubular mitochondria in wild-type cells, mitochondria in gem1Δ cells are present in clusters containing fully resolved individual organelles (Figure 3A–D and Videos 1–4). Importantly, fully separate and adjacent mitochondria interacted with the same ER segment (Figure 3B,C, B₂ and C₁, ER–mitochondrial contact sites in red), suggesting that after division, resolved mitochondria remain tethered via this shared ER segment.

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Quantitative analysis of an equivalent mitochondrial surface area in wild-type and gem1Δ cells (approximately 5 μm²) revealed that gem1Δ cells harbor a significantly greater number of ER–mitochondria contacts than wild-type cells, which are also more clustered as compared to wild-type contacts (Figure 3—figure supplement 1). The surface area of individual ER–mitochondria contacts was, on average, smaller in gem1Δ cells as compared to wild-type cells (Figure 3E and Figure 3—figure supplement 1C). However, the total ER–mitochondria contact surface was approximately three times greater than that observed for wild-type mitochondria (Figure 3F). These findings are consistent with fluorescence data demonstrating that, in comparison to wild-type cells, ER tubules in gem1Δ cells were observed associated in a relatively stable manner with mitochondrial constriction sites (Figure 2C). In addition, our EM analysis suggests that the reported larger ERMES foci observed by fluorescence imaging in gem1Δ cells may represent multiple unresolved ER–mitochondria contacts (Kornmann et al., 2011) (Figure 3—figure supplement 1D). The average diameter of gem1Δ mitochondria, measured along their short axis, was also greater than that of wild-type cells (Figure 3—figure supplement 1E), consistent with our fluorescence imaging analysis (Figure 2). However, the average diameter of ER-associated mitochondrial constriction sites in wild-type and gem1Δ cells were similar (Figure 3—figure supplement 1F), consistent with our light imaging data demonstrating that Dnm1 is still efficiently targeted to mitochondrial constriction sites in gem1Δ cells (Figure 2C). Together, these data suggest that Gem1 functions downstream of ER-associated mitochondrial constriction and division as a negative regulator of ER–mitochondria contacts, which is required for the efficient resolution and spatial distribution of mitochondria following division.

To further investigate the functional significance of these observations, we explored the idea that ERMD serves to position division sites adjacent to actively segregating mitochondrial nucleoids, which is based on the observation that ERMES is spatially linked to actively replicating mitochondrial nucleoids (Hobbs et al., 2001; Meeusen and Nunnari, 2003). To visualize nucleoids, we created a functional FP fusion to the nucleoid component, Yme2 (Figure 4 and Figure 4—figure supplements 1 and 2), an integral mitochondrial inner membrane protein with homology to exonucleases whose deletion alters nucleoid structure and copy number (Hanekamp and Thorsness, 1996, 1999; Park et al., 2006). Consistent with the previously published work, we observed that over time a subset of Yme2-GFP–labeled nucleoids persistently localize adjacent to ERMES foci, marked by Mdm34-tdTomato (Figure 4A). In addition, ERMES-linked nucleoids were associated with mitochondrial division events (Figure 4A). To characterize the behavior of yeast nucleoids at division sites, we simultaneously imaged mitochondria (mito-dsRed) and nucleoids (Yme2-GFP). Quantitative analysis indicated that mitochondrial nucleoids are associated with over 80% of mitochondrial division events (Figure 4B,C). Temporal analysis of Yme2-GFP–labeled nucleoids positioned at future sites of mitochondrial division indicated that they exhibit short-ranged oscillatory movements and often rapidly segregate and recoalesce prior to mitochondrial division (Figure 4B and Figure 4—figure supplement 3). Following mitochondrial division, a majority of Yme2-GFP foci were observed at the ends of both newly generated mitochondrial tips (Figure 4B,C and Figure 4—figure supplement 3). However, we also observed division events that resulted in a nucleoid in only one of the newly generated mitochondrial tips, indicating that division and nucleoid segregation are not obligatorily linked (Figure 4C and Figure 4—figure supplement 3). Consistent with a role of ERMES in nucleoid positioning, published data indicate that deletion of core ERMES components disrupts nucleoid structure (Burgess et al., 1994; Youngman et al., 2004). In contrast, we observed that nucleoids, as labeled by Yme2-GFP, maintained their discrete focal localization in gem1Δ cells, similar to wild-type cells (Figure 4D). In addition, similar to ERMES distribution, Yme2-GFP foci were observed associated with stable mitochondrial constrictions in gem1Δ cells (Figure 4D), consistent with the idea that the spatial resolution of ER–mitochondria contacts following division is fundamentally important for nucleoid distribution. Thus, our findings suggest that ERMD functions to position division sites in proximity to nucleoids to increase the probability of their distribution upon mitochondrial division.

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Here, we show that ERMES and the Miro GTPase Gem1 function in the process of ERMD, which serves to couple the segregation of mitochondria and mtDNA in cells. Specifically, our data support a model in which ERMES creates ER–mitochondria contacts along the mitochondrial network that serve to link actively replicating nucleoids to mitochondrial division and the subsequent Gem1-dependent spatial resolution of newly generated mitochondrial tips. Such a mechanism is likely required for efficient mtDNA distribution throughout the cell in addition to nucleoid segregation per se, as long-range movement of nucleoids within the organelle is limited (Nunnari et al., 1997; Okamoto et al., 1998). In mammalian cells, nucleoids are similarly localized at mitochondrial division sites and mitochondrial tips, suggesting that ERMD also plays this fundamental role in humans (Garrido et al., 2003; Iborra et al., 2004). In this context, although the Miro GTPase family is highly conserved in eukaryotes, Miro orthologs are not found in organisms that lack mtDNA, such as Giardia intestinalis and Trichomonas vaginalis, and possess mitochondrial-related mitosomes or hydrogenosomes, respectively (Vlahou et al., 2011). This correlation is consistent with a fundamental role of Miro in mtDNA segregation. Also consistent with this view, gem1Δ cells lose mitochondrial DNA at a significantly higher frequency than wild-type cells (Frederick et al., 2004).

The mechanism of mitochondrial division site placement is apparently divergent from that utilized by ancestral bacteria, where division sites are determined in part by a nucleoid occlusion mechanism, which prevents cell division in the vicinity of the bacterial chromosome (Wu and Errington, 2012). However, we currently lack an understanding of what drives mitochondrial nucleoid segregation and of the mechanism underlying the spatial coupling of ER–mitochondria contact sites and nucleoids. Thus, alternative mitochondrial-specific mechanisms may exist to coordinate the timing of nucleoid replication and segregation with mitochondrial division.

In ERMD, mitochondrial constrictions observed at the sites of ER–mitochondria contact, where the ER likely wraps around a mitochondrial tubule, are independent of the mitochondrial division dynamin (Friedman et al., 2011). Our data are consistent with the model in which ERMES functions early in ERMD to mediate the biogenesis of this specialized region of ER–mitochondria contact. Thus, it is tempting to speculate that the ERMES complex functions to generate the ER tubules associated with constriction and/or to actively produce mitochondrial constriction at the sites of contact. ERMES has also been implicated as a bridge between mitochondria and the actomyosin network and thus may function at division sites to coordinate the recruitment of cytoskeletal and motor proteins, which could generate force required for mitochondrial constriction and/or distribution following division (Boldogh et al., 1998, 2003). Indeed, a recent study suggests that the actin cytoskeleton may be involved in mitochondrial division at ER–mitochondria contacts in mammalian cells (Korobova et al., 2013).

In contrast to ERMES, our data indicate that Gem1 functions relatively late in ERMD to promote the physical separation of mitochondrial tips generated by membrane scission. Specifically, our data point to a role for Gem1 post-scission in the negative regulation of ER–mitochondria contacts to facilitate the resolution of mitochondrial tips. Evidence from higher eukaryotes suggests that the Gem1 ortholog, Miro1/2, functions in mitochondrial distribution by connecting mitochondria directly to a kinesin-1 adaptor protein Milton/TRAK to enable the microtubule based transport of mitochondria (Guo et al., 2005; Fransson et al., 2006; Glater et al., 2006; Wang and Schwarz, 2009; Misko et al., 2010; Wang et al., 2011). Analogously, Gem1 in yeast may also function to negatively regulate ER–mitochondria contacts by facilitating the recruitment of motility factors to mitochondrial tips following division. However, while the Miro GTPase family is remarkably conserved (Vlahou et al., 2011), the mechanisms of mitochondrial transport are divergent in eukaryotes. In many fungi, including budding yeast, mitochondrial distribution is actin dependent (Hermann et al., 1997; Simon et al., 1997; Boldogh et al., 1998, 2003), and in Dictyostelium, Miro is not required for microtubule-dependent mitochondrial transport (Vlahou et al., 2011). Thus, alternatively, Gem1 may function in ERMD more directly to regulate the physical link between mitochondria and the ER via ERMES. Consistent with this idea, Gem1 associates with ERMES foci, and, in the absence of Gem1, it has been reported that ERMES forms fewer and larger foci per cell (Kornmann et al., 2011). This model of Gem1 function is also supported by the observation that mutations in the first GTPase domain of Gem1 abolish both Gem1’s function in ERMD and its association with ERMES (Frederick et al., 2004; Kornmann et al., 2011; Koshiba et al., 2011). In addition, in mammalian cells, Miro1 is found at ER–mitochondria contacts (Kornmann et al., 2011). In this context, the defect in mitochondrial segregation observed in gem1Δ cells at ER-associated mitochondrial constriction sites could result from the ER physically hindering mitochondrial motility. In this case, it is also possible that the mitochondrial motility defects in higher eukaryotes caused by Miro1/2 dysfunction are a secondary consequence of enhanced ER–mitochondria contacts.

1. 1. Boldogh I
   2. Vojtov N
   3. Karmon S
   4. Pon LA

   (1998) Interaction between mitochondria and the actin cytoskeleton in budding yeast requires two integral mitochondrial outer membrane proteins, Mmm1p and Mdm10p

   J Cell Biol 141:1371–1381.

   https://doi.org/10.1083/jcb.141.6.1371

   • Google Scholar
2. 1. Boldogh IR
   2. Nowakowski DW
   3. Yang HC
   4. Chung H
   5. Karmon S
   6. Royes P

   et al. (2003) A protein complex containing Mdm10p, Mdm12p, and Mmm1p links mitochondrial membranes and DNA to the cytoskeleton-based segregation machinery

   Mol Biol Cell 14:4618–4627.

   https://doi.org/10.1091/mbc.E03-04-0225

   • Google Scholar
3. 1. Bossy B
   2. Petrilli A
   3. Klinglmayr E
   4. Chen J
   5. Lutz-Meindl U
   6. Knott AB

   et al. (2010) S-Nitrosylation of DRP1 does not affect enzymatic activity and is not specific to Alzheimer’s disease

   J Alzheimers Dis 20:S513–S526.

   https://doi.org/10.3233/JAD-2010-100552

   • Google Scholar
4. 1. Boulanger J
   2. Kervrann C
   3. Bouthemy P
   4. Elbau P
   5. Sibarita JB
   6. Salamero J

   (2010) Patch-based nonlocal functional for denoising fluorescence microscopy image sequences

   IEEE Trans Med Imaging 29:442–454.

   https://doi.org/10.1109/TMI.2009.2033991

   • Google Scholar
5. 1. Brachmann CB
   2. Davies A
   3. Cost GJ
   4. Caputo E
   5. LI J
   6. Hieter P

   et al. (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications

   Yeast 14:115–132.

   https://doi.org/10.1002/(SICI)1097-0061(19980130)14:2<115::AID-YEA204>3.0.CO;2-2

   • Google Scholar
6. 1. Burgess SM
   2. Delannoy M
   3. Jensen RE

   (1994) MMM1 encodes a mitochondrial outer membrane protein essential for establishing and maintaining the structure of yeast mitochondria

   J Cell Biol 126:1375–1391.

   https://doi.org/10.1083/jcb.126.6.1375

   • Google Scholar
7. 1. Carlton PM
   2. Boulanger J
   3. Kervrann C
   4. Sibarita JB
   5. Salamero J
   6. Gordon-Messer S

   et al. (2010) Fast live simultaneous multiwavelength four-dimensional optical microscopy

   Proc Natl Acad Sci USA 107:16016–16022.

   https://doi.org/10.1073/pnas.1004037107

   • Google Scholar
8. 1. de Brito OM
   2. Scorrano L

   (2008) Mitofusin 2 tethers endoplasmic reticulum to mitochondria

   Nature 456:605–610.

   https://doi.org/10.1038/nature07534

   • Google Scholar
9. 1. Fransson S
   2. Ruusala A
   3. Aspenstrom P

   (2006) The atypical Rho GTPases Miro-1 and Miro-2 have essential roles in mitochondrial trafficking

   Biochem Biophys Res Commun 344:500–510.

   https://doi.org/10.1016/j.bbrc.2006.03.163

   • Google Scholar
10. 1. Frederick RL
    2. McCaffery JM
    3. Cunningham KW
    4. Okamoto K
    5. Shaw JM

    (2004) Yeast Miro GTPase, Gem1p, regulates mitochondrial morphology via a novel pathway

    J Cell Biol 167:87–98.

    https://doi.org/10.1083/jcb.200405100

    • Google Scholar
11. 1. Friedman JR
    2. Lackner LL
    3. West M
    4. Dibenedetto JR
    5. Nunnari J
    6. Voeltz GK

    (2011) ER tubules mark sites of mitochondrial division

    Science 334:358–362.

    https://doi.org/10.1126/science.1207385

    • Google Scholar
12. 1. Garrido N
    2. Griparic L
    3. Jokitalo E
    4. Wartiovaara J
    5. Van der Bliek AM
    6. Spelbrink JN

    (2003) Composition and dynamics of human mitochondrial nucleoids

    Mol Biol Cell 14:1583–1596.

    https://doi.org/10.1091/mbc.E02-07-0399

    • Google Scholar
13. 1. Giddings TH

    (2003)

    Freeze-substitution protocols for improved visualization of membranes in high-pressure frozen samples

    J Microsc 212:53–61.

    • Google Scholar
14. 1. Glater EE
    2. Megeath LJ
    3. Stowers RS
    4. Schwarz TL

    (2006) Axonal transport of mitochondria requires milton to recruit kinesin heavy chain and is light chain independent

    J Cell Biol 173:545–557.

    https://doi.org/10.1083/jcb.200601067

    • Google Scholar
15. 1. Guo X
    2. Macleod GT
    3. Wellington A
    4. Hu F
    5. Panchumarthi S
    6. Schoenfield M

    et al. (2005) The GTPase dMiro is required for axonal transport of mitochondria to Drosophila synapses

    Neuron 47:379–393.

    https://doi.org/10.1016/j.neuron.2005.06.027

    • Google Scholar
16. 1. Hanekamp T
    2. Thorsness PE

    (1996)

    Inactivation of YME2/RNA12, which encodes an integral inner mitochondrial membrane protein, causes increased escape of DNA from mitochondria to the nucleus in Saccharomyces cerevisiae

    Mol Cell Biol 16:2764–2771.

    • Google Scholar
17. 1. Hanekamp T
    2. Thorsness PE

    (1999) YNT20, a bypass suppressor of yme1 yme2, encodes a putative 3′-5′ exonuclease localized in mitochondria of Saccharomyces cerevisiae

    Curr Genet 34:438–448.

    https://doi.org/10.1007/s002940050418

    • Google Scholar
18. 1. Hanekamp T
    2. Thorsness MK
    3. Rebbapragada I
    4. Fisher EM
    5. Seebart C
    6. Darland MR

    et al. (2002)

    Maintenance of mitochondrial morphology is linked to maintenance of the mitochondrial genome in Saccharomyces cerevisiae

    Genetics 162:1147–1156.

    • Google Scholar
19. 1. Hermann GJ
    2. King EJ
    3. Shaw JM

    (1997) The yeast gene, MDM20, is necessary for mitochondrial inheritance and organization of the actin cytoskeleton

    J Cell Biol 137:141–153.

    https://doi.org/10.1083/jcb.137.1.141

    • Google Scholar
20. 1. Hobbs AE
    2. Srinivasan M
    3. Mccaffery JM
    4. Jensen RE

    (2001) Mmm1p, a mitochondrial outer membrane protein, is connected to mitochondrial DNA (mtDNA) nucleoids and required for mtDNA stability

    J Cell Biol 152:401–410.

    https://doi.org/10.1083/jcb.152.2.401

    • Google Scholar
21. 1. Huh WK
    2. Falvo JV
    3. Gerke LC
    4. Carroll AS
    5. Howson RW
    6. Weissman JS

    et al. (2003) Global analysis of protein localization in budding yeast

    Nature 425:686–691.

    https://doi.org/10.1038/nature02026

    • Google Scholar
22. 1. Iborra FJ
    2. Kimura H
    3. Cook PR

    (2004) The functional organization of mitochondrial genomes in human cells

    BMC Biol 2:9.

    https://doi.org/10.1186/1741-7007-2-9

    • Google Scholar
23. 1. Ingerman E
    2. Perkins EM
    3. Marino M
    4. Mears JA
    5. McCaffery JM
    6. Hinshaw JE

    et al. (2005) Dnm1 forms spirals that are structurally tailored to fit mitochondria

    J Cell Biol 170:1021–1027.

    https://doi.org/10.1083/jcb.200506078

    • Google Scholar
24. 1. Janke C
    2. Magiera MM
    3. Rathfelder N
    4. Taxis C
    5. Reber S
    6. Maekawa H

    et al. (2004) A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes

    Yeast 21:947–962.

    https://doi.org/10.1002/yea.1142

    • Google Scholar
25. 1. Kornmann B
    2. Currie E
    3. Collins SR
    4. Schuldiner M
    5. Nunnari J
    6. Weissman JS

    et al. (2009) An ER-mitochondria tethering complex revealed by a synthetic biology screen

    Science 325:477–481.

    https://doi.org/10.1126/science.1175088

    • Google Scholar
26. 1. Kornmann B
    2. Osman C
    3. Walter P

    (2011) The conserved GTPase Gem1 regulates endoplasmic reticulum–mitochondria connections

    Proc Natl Acad Sci USA 108:14151–14156.

    https://doi.org/10.1073/pnas.1111314108

    • Google Scholar
27. 1. Korobova F
    2. Ramabhadran V
    3. Higgs HN

    (2013) An actin-dependent step in mitochondrial fission mediated by the ER-associated formin INF2

    Science 339:464–467.

    https://doi.org/10.1126/science.1228360

    • Google Scholar
28. 1. Koshiba T
    2. Holman HA
    3. Kubara K
    4. Yasukawa K
    5. Kawabata S
    6. Okamoto K

    et al. (2011) Structure-function analysis of the yeast mitochondrial Rho GTPase, Gem1p: implications for mitochondrial inheritance

    J Biol Chem 286:354–362.

    https://doi.org/10.1074/jbc.M110.180034

    • Google Scholar
29. 1. Kremer JR
    2. Mastronarde DN
    3. McIntosh JR

    (1996) Computer visualization of three-dimensional image data using IMOD

    J Struct Biol 116:71–76.

    https://doi.org/10.1006/jsbi.1996.0013

    • Google Scholar
30. 1. Lackner LL
    2. Horner JS
    3. Nunnari J

    (2009) Mechanistic analysis of a dynamin effector

    Science 325:874–877.

    https://doi.org/10.1126/science.1176921

    • Google Scholar
31. 1. Lackner LL
    2. Nunnari JM

    (2009) The molecular mechanism and cellular functions of mitochondrial division

    Biochim Biophys Acta 1792:1138–1144.

    https://doi.org/10.1016/j.bbadis.2008.11.011

    • Google Scholar
32. 1. Mastronarde DN

    (1997) Dual-axis tomography: an approach with alignment methods that preserve resolution

    J Struct Biol 120:343–352.

    https://doi.org/10.1006/jsbi.1997.3919

    • Google Scholar
33. 1. Mears JA
    2. Lackner LL
    3. Fang S
    4. Ingerman E
    5. Nunnari J
    6. Hinshaw JE

    (2011) Conformational changes in Dnm1 support a contractile mechanism for mitochondrial fission

    Nat Struct Mol Biol 18:20–26.

    https://doi.org/10.1038/nsmb.1949

    • Google Scholar
34. 1. Meeusen S
    2. Nunnari J

    (2003) Evidence for a two membrane-spanning autonomous mitochondrial DNA replisome

    J Cell Biol 163:503–510.

    https://doi.org/10.1083/jcb.200304040

    • Google Scholar
35. 1. Misko A
    2. Jiang S
    3. Wegorzewska I
    4. Milbrandt J
    5. Baloh RH

    (2010) Mitofusin 2 is necessary for transport of axonal mitochondria and interacts with the Miro/Milton complex

    J Neurosci 30:4232–4240.

    https://doi.org/10.1523/JNEUROSCI.6248-09.2010

    • Google Scholar
36. 1. Nickerson DP
    2. West M
    3. Henry R
    4. Odorizzi G

    (2010) Regulators of Vps4 ATPase activity at endosomes differentially influence the size and rate of formation of intralumenal vesicles

    Mol Biol Cell 21:1023–1032.

    https://doi.org/10.1091/mbc.E09-09-0776

    • Google Scholar
37. 1. Nunnari J
    2. Marshall WF
    3. Straight A
    4. Murray A
    5. Sedat JW
    6. Walter P

    (1997)

    Mitochondrial transmission during mating in Saccharomyces cerevisiae is determined by mitochondrial fusion and fission and the intramitochondrial segregation of mitochondrial DNA

    Mol Biol Cell 8:1233–1242.

    • Google Scholar
38. 1. Okamoto K
    2. Perlman PS
    3. Butow RA

    (1998) The sorting of mitochondrial DNA and mitochondrial proteins in zygotes: preferential transmission of mitochondrial DNA to the medial bud

    J Cell Biol 142:613–623.

    https://doi.org/10.1083/jcb.142.3.613

    • Google Scholar
39. 1. Park S
    2. Hanekamp T
    3. Thorsness MK
    4. Thorsness PE

    (2006) Yme2p is a mediator of nucleoid structure and number in mitochondria of the yeast Saccharomyces cerevisiae

    Curr Genet 50:173–182.

    https://doi.org/10.1007/s00294-006-0087-9

    • Google Scholar
40. 1. Rossanese OW
    2. Reinke CA
    3. Bevis BJ
    4. Hammond AT
    5. Sears IB
    6. O’Connor J

    et al. (2001) A role for actin, Cdc1p, and Myo2p in the inheritance of late golgi elements in Saccharomyces cerevisiae

    J Cell Biol 153:47–62.

    https://doi.org/10.1083/jcb.153.1.47

    • Google Scholar
41. 1. Rothstein RJ

    (1983) One-step gene disruption in yeast

    Methods Enzymol 101:202–211.

    https://doi.org/10.1016/0076-6879(83)01015-0

    • Google Scholar
42. 1. Shaner NC
    2. Campbell RE
    3. Steinbach PA
    4. Giepmans BN
    5. Palmer AE
    6. Tsien RY

    (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein

    Nat Biotechnol 22:1567–1572.

    https://doi.org/10.1038/nbt1037

    • Google Scholar
43. 1. Sheff MA
    2. Thorn KS

    (2004) Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae

    Yeast 21:661–670.

    https://doi.org/10.1002/yea.1130

    • Google Scholar
44. 1. Simon VR
    2. Karmon SL
    3. Pon LA

    (1997) Mitochondrial inheritance: cell cycle and actin cable dependence of polarized mitochondrial movements in Saccharomyces cerevisiae

    Cell Motil Cytoskeleton 37:199–210.

    https://doi.org/10.1002/(SICI)1097-0169(1997)37:3<199::AID-CM2>3.0.CO;2-2

    • Google Scholar
45. 1. Stroud DA
    2. Oeljeklaus S
    3. Wiese S
    4. Bohnert M
    5. Lewandrowski U
    6. Sickmann A

    et al. (2011) Composition and topology of the endoplasmic reticulum-mitochondria encounter structure

    J Mol Biol 413:743–750.

    https://doi.org/10.1016/j.jmb.2011.09.012

    • Google Scholar
46. 1. Toulmay A
    2. Prinz WA

    (2012) A conserved membrane-binding domain targets proteins to organelle contact sites

    J Cell Sci 125:49–58.

    https://doi.org/10.1242/jcs.085118

    • Google Scholar
47. 1. Vlahou G
    2. Elias M
    3. von Kleist-Retzow JC
    4. Wiesner RJ
    5. Rivero F

    (2011) The Ras related GTPase Miro is not required for mitochondrial transport in Dictyostelium discoideum

    Eur J Cell Biol 90:342–355.

    https://doi.org/10.1016/j.ejcb.2010.10.012

    • Google Scholar
48. 1. Voss C
    2. Lahiri S
    3. Young BP
    4. Loewen CJ
    5. Prinz WA

    (2012) ER-shaping proteins facilitate lipid exchange between the ER and mitochondria in S. cerevisiae

    J Cell Sci 125:4791–4799.

    https://doi.org/10.1242/jcs.105635

    • Google Scholar
49. 1. Wang X
    2. Schwarz TL

    (2009) The mechanism of Ca²⁺ -dependent regulation of kinesin-mediated mitochondrial motility

    Cell 136:163–174.

    https://doi.org/10.1016/j.cell.2008.11.046

    • Google Scholar
50. 1. Wang X
    2. Winter D
    3. Ashrafi G
    4. Schlehe J
    5. Wong YL
    6. Selkoe D

    et al. (2011) PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility

    Cell 147:893–906.

    https://doi.org/10.1016/j.cell.2011.10.018

    • Google Scholar
51. 1. West M
    2. Zurek N
    3. Hoenger A
    4. Voeltz GK

    (2011) A three-dimenstional analysis of yeast ER structure reveals how ER domains are organized by membrane curvature

    J Cell Biol 193:333–346.

    https://doi.org/10.1083/jcb.201011039

    • Google Scholar
52. 1. Westermann B
    2. Neupert W

    (2000) Mitochondria-targeted green fluorescent proteins: convenient tools for the study of organelle biogenesis in Saccharomyces cerevisiae

    Yeast 16:1421–1427.

    https://doi.org/10.1002/1097-0061(200011)16:15<1421::AID-YEA624>3.0.CO;2-U

    • Google Scholar
53. 1. Wu LJ
    2. Errington J

    (2012) Nucleoid occlusion and bacterial cell division

    Nat Rev Microbiol 10:8–12.

    https://doi.org/10.1038/nrmicro2671

    • Google Scholar
54. 1. Youngman MJ
    2. Hobbs AE
    3. Burgess SM
    4. Srinivasan M
    5. Jensen RE

    (2004) Mmm2p, a mitochondrial outer membrane protein required for yeast mitochondrial shape and maintenance of mtDNA nucleoids

    J Cell Biol 164:677–688.

    https://doi.org/10.1083/jcb.200308012

    • Google Scholar

Author details

1. Andrew Murley

   Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States

   Contribution

   AM, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

2. Laura L Lackner

   Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States

   Contribution

   LLL, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Christof Osman

   Competing interests

   No competing interests declared.

3. Christof Osman

   Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States

   Contribution

   CO, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Laura L Lackner

   Competing interests

   No competing interests declared.

4. Matthew West

   Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Boulder, United States

   Contribution

   MW, Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

5. Gia K Voeltz

   Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Boulder, United States

   Contribution

   GKV, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

6. Peter Walter

   1. Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
   2. Howard Hughes Medical Institute, University of California, San Francisco, United States

   Contribution

   PW, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

7. Jodi Nunnari

   Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States

   Contribution

   JN, Conception and design, Drafting or revising the article

   For correspondence

   jmnunnari@ucdavis.edu

   Competing interests

   JN, Reviewing editor, eLife.

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