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The dynamin-related protein 1 (Drp1) and its homologs in various eukaryotes are essential to maintain mitochondrial morphology and regulate mitochondrial division. Several mutations in different domains of Drp1 have been reported, which result in debilitating conditions. Four such disease-causing mutations of the middle domain of Drp1 were mimicked in the yeast dynamin-related GTPase (Dnm1) and were characterized in this study. Mitochondrial morphology and protein function were observed to be altered to a variable extent in cells expressing the mutated variants of Dnm1. Several aspects related to the protein such as punctate formation, localization to mitochondria, dynamic behavior and structure were analyzed by microscopy, biochemical studies and molecular dynamics simulations. Significant effects on the protein structure and function were observed in cells expressing A430D and G397D mutations. Overall, our data provide insight into the molecular and cellular alterations resulting from middle domain mutations in Dnm1.

Mitochondrial division and fusion play critical roles in maintaining functional mitochondria. Fzo1 is an outer mitochondrial membrane GTPase that played an essential role in mitochondrial fusion in budding yeast Saccharomyces cerevisiae. Here, we report the characterization of the Schizosaccharomyces pombe homologue of S. cerevisiae Fzo1p, Fzo1. Disruption of the fzo1 gene in S. pombe results in a fragmented mitochondrial morphology and a dramatically reduced growth on glycerol medium phenotype, indicating that deletion of fzo1 compromises respiratory function. Fluorescence microscopy shows that Fzo1p is located in the mitochondria. Overexpressing Fzo1 from a heterologous promoter induces mitochondrial aggregation. We also find that dnm1 mutations could both block mitochondrial fragmentation and rescue respiration growth defect in Δfzo1 single mutant cells. Our results proposed that a genetic interaction between fzo1 and a balance between division- and fusion-controlled mitochondrial shape and function in S. pombe. This study represents the first report of Fzo1 mediator of mitochondrial fusion in S. pombe.

Mitochondria are dynamic organelles that undergo cycles of fission and fusion. The major mitochondrial fission protein is dynamin-related Drp1 GTPase (Dnm1 in yeast). The effects of Dnm1 gene deletion on cell dynamics and energy metabolism during mitosis were studied in Schizosaccharomyces pombe. Dnm1 gene deletion can lead to slow growth, abnormal sporulation, abnormal number and length of interphase microtubules of Schizosaccharomyces pombe. The deletion of Dnm1 gene can also affect the spindle growth speed and growth time of metaphase and anaphase, and affect the spindle fluorescence intensity of prophase and metaphase. At the same time, the structure and dynamics of the spindle microtubules of Dnm1Δ are also different. The statistics of spindle length showed that there was delayed spindle fracture in Dnm1Δ Cells. Two different chromosome behaviors, normal and lagged, were observed by living cell imaging. The analysis of coenzymes, intermediates and energy in energy metabolism showed that there were some abnormalities after Dnm1 gene deletion, including coenzyme defects, intermediate metabolite defects and ATP production defects.

Mitochondria and peroxisomes are ubiquitous subcellular organelles that are highly dynamic and possess a high degree of plasticity. These organelles proliferate through division of pre-existing organelles. Studies on yeast, mammalian cells, and unicellular algae have led to a surprising finding that mitochondria and peroxisomes share the components of their division machineries. At the heart of the mitochondrial and peroxisomal division machineries is a GTPase dynamin-like protein, Dnm1/Drp1, which forms a contractile ring around the neck of the dividing organelles. During division, Dnm1/Drp1 functions as a motor protein and constricts the membrane. This mechanochemical work is achieved by utilizing energy from GTP hydrolysis. Over the last two decades, studies have focused on the structure and assembly of Dnm1/Drp1 molecules around the neck. However, the regulation of GTP during the division of mitochondrion and peroxisome is not well understood. Here, we review the current understanding of Dnm1/Drp1-mediated divisions of mitochondria and peroxisomes, exploring the mechanisms of GTP regulation during the Dnm1/Drp1 function, and provide new perspectives on their potential contribution to mitochondrial and peroxisomal biogenesis.

Mitochondrial fission shows potential as a therapeutic target in non-infectious human diseases. The compound mdivi-1 was identified as a mitochondrial fission inhibitor that acts against the evolutionarily conserved mitochondrial fission GTPase Dnm1/Drp1, and shows promising data in pre-clinical models of human pathologies. Two recent studies, however, found no evidence that mdivi-1 acts as a mitochondrial fission inhibitor and proposed other mechanisms. In mammalian cells, Bordt et al. showed that mdivi-1 inhibits complex I in mitochondria (Dev Cell 40:583, 2017). In a second study, we have recently demonstrated that mdivi-1 does not trigger a mitochondrial morphology change in the human yeast pathogen Candida albicans, but impacts on endogenous nitric oxide (NO) levels and inhibits the key virulence property of hyphal formation (Koch et al., Cell Rep 25:2244, 2018). Here we discuss recent insights into mdivi-1’s action in pathogenic fungi and the potential and challenges for repurposing it as an anti-infective. We also outline recent findings on the roles of mitochondrial fission in human and plant fungal pathogens, with the goal of starting the conversation on whether the research field of fungal pathogenesis can benefit from efforts in other disease areas aimed at developing therapeutic inhibitors of mitochondrial division.

Summary Dynamic processes such as fusion, fission, and trafficking are important in the regulation of cellular organelles, with an abundant literature focused on mitochondria. Mitochondrial dynamics not only help shape its network within cells but also are involved in the modulation of respiration and integrity. Disruptions of mitochondrial dynamics are associated with neurodegenerative disorders. Although proteins that directly bind mitochondria to promote membrane fusion/fission have been studied intensively, machineries that regulate dynamic mitochondrial processes remain to be explored. We have identified an interaction between the mitochondrial fission GTPase Dnm1/DRP1 and the actin-regulatory protein Srv2/CAP at mitochondria. Deletion of Srv2 causes elongated-hyperfused mitochondria and reduces the reserved respiration capacity in yeast cells. Our results further demonstrate that the irregular network morphology in Δsrv2 cells derives from disrupted actin assembly at mitochondria. We suggest that Srv2 functions as a pro-fission factor in shaping mitochondrial dynamics and regulating activity through its actin-regulatory effects., Graphical Abstract, Highlights • Srv2 interacts with fission protein Dnm1 on mitochondria in yeast cells • Srv2 deletion causes an irregular, hyperfused-elongated mitochondrial network • The irregular network derives from loss of Srv2-mediated actin assembly at mitochondria • Srv2 modulates both mitochondrial dynamics and activity, Molecular Biology; Cell Biology; Functional Aspects of Cell Biology

Dnm1 and Fis1 are prototypical proteins that regulate yeast mitochondrial morphology by controlling fission, the dysregulation of which can result in developmental disorders and neurodegenerative diseases in humans. Loss of Dnm1 blocks the formation of fission complexes and leads to elongated mitochondria in the form of interconnected networks, while overproduction of Dnm1 results in excessive mitochondrial fragmentation. In the current model, Dnm1 is essentially a GTP hydrolysis-driven molecular motor that self-assembles into ring-like oligomeric structures that encircle and pinch the outer mitochondrial membrane at sites of fission. In this work, we use machine learning and synchrotron small-angle X-ray scattering (SAXS) to investigate whether the motor Dnm1 can synergistically facilitate mitochondrial fission by membrane remodeling. A support vector machine (SVM)-based classifier trained to detect sequences with membrane-restructuring activity identifies a helical Dnm1 domain capable of generating negative Gaussian curvature (NGC), the type of saddle-shaped local surface curvature found on scission necks during fission events. Furthermore, this domain is highly conserved in Dnm1 homologues with fission activity. Synchrotron SAXS measurements reveal that Dnm1 restructures membranes into phases rich in NGC, and is capable of inducing a fission neck with a diameter of 12.6 nm. Through in silico mutational analysis, we find that the helical Dnm1 domain is locally optimized for membrane curvature generation, and phylogenetic analysis suggests that dynamin superfamily proteins that are close relatives of human dynamin Dyn1 have evolved the capacity to restructure membranes via the induction of curvature mitochondrial fission. In addition, we observe that Fis1, an adaptor protein, is able to inhibit the pro-fission membrane activity of Dnm1, which points to the antagonistic roles of the two proteins in the regulation of mitochondrial fission., Yeast Dnm1 (human Drp1) is a molecular motor that can remodel membranes to promote scission necks during mitochondrial fission.

Mitochondria undergo morphological and dynamic changes in response to environmental stresses. Few studies have focused on addressing mitochondrial remodeling under stress. Using the fission yeast Schizosaccharomyces pombe as a model organism, here we investigated mitochondrial remodeling under glucose starvation. We employed live-cell microscopy to monitor mitochondrial morphology and dynamics of cells in profusion chambers under glucose starvation. Our results revealed that mitochondria fragment within minutes after glucose starvation and that the dynamin GTPase Dnm1 is required for promoting mitochondrial fragmentation. Moreover, we found that glucose starvation enhances Dnm1 localization to mitochondria and increases the frequency of mitochondrial fission but decreases PKA activity. We further demonstrate that low PKA activity enhances glucose starvation–induced mitochondrial fragmentation, whereas high PKA activity confers resistance to glucose starvation–induced mitochondrial fragmentation. Moreover, we observed that AMP-activated protein kinase is not involved in regulating mitochondrial fragmentation under glucose starvation. Of note, glucose starvation–induced mitochondrial fragmentation was associated with enhanced reactive oxygen species production. Our work provides detailed mechanistic insights into mitochondrial remodeling in response to glucose starvation.

Summary Magnaporthe oryzae causes blast disease, which is one of the most devastating infections in rice and several important cereal crops. Magnaporthe oryzae needs to coordinate gene regulation, morphological changes, nutrient acquisition and host evasion in order to invade and proliferate within the plant tissues. Thus far, the molecular mechanisms underlying the regulation of invasive growth in planta have remained largely unknown. We identified a precise filamentous‐punctate‐filamentous cycle in mitochondrial morphology during Magnaporthe–rice interaction. Interestingly, disruption of such mitochondrial dynamics by deletion of genes regulating either the mitochondrial fusion (MoFzo1) or fission (MoDnm1) machinery, or inhibition of mitochondrial fission using Mdivi‐1 caused significant reduction in M. oryzae pathogenicity. Furthermore, exogenous carbon source(s) but not antioxidant treatment delayed such mitochondrial dynamics/transition during invasive growth. In contrast, carbon starvation induced the breakdown of the mitochondrial network and led to more punctate mitochondria in vitro. Such nutrient‐based regulation of organellar dynamics preceded MoAtg24‐mediated mitophagy, which was found to be essential for proper biotrophic development and invasive growth in planta. We propose that precise mitochondrial dynamics and mitophagy occur during the transition from biotrophy to necrotrophy and are required for proper induction and establishment of the blast disease in rice.

TDP-43 is a multi-functional ribonucleoprotein that is also found deposited as hyper-phosphorylated and ubiquitinated TDP-43 inclusions in the brain and spinal cord of the patients of the motor neuron diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Till date, how the cell death ensues is not fully deciphered although several molecular mechanisms of the TDP-43 toxicity such as impairments of endocytosis and chromatin remodelling, mis-regulations of autophagy and proteasome function, mis-localization to the mitochondria and generation of oxidative stress etc., have been proposed. A predominantly nuclear protein, Cyclin C, can regulate the oxidative stress response by affecting the transcription of stress response genes and also by translocation to the cytoplasm for the activation of the mitochondrial fragmentation-dependent cell death pathway. Using the well-established yeast model of TDP-43 aggregation and toxicity, we examined here whether upon TDP-43 aggregation, the cell survival depends on the presence of theCNC1gene that encodes Cyclin C protein or other genes that encode proteins that function in conjunction with Cyclin C, such as theDNM1, FIS1andMED13genes. We found that the TDP-43 toxicity is significantly reduced in the yeast deleted for theCNC1orDNM1genes. Importantly, the rescue of TDP-43 toxicity in these yeast deletion backgrounds required the presence of functional mitochondria. Also, the deletion ofYBH3gene, which encodes for a protein involved in the mitochondria-dependent apoptosis, also reduced the TDP-43 toxicity. Furthermore, Cyclin C-YFP was observed to localize from the nucleus to the cytoplasm in response to the TDP-43 co-expression. Also, this cytoplasmic localization of Cyclin C was prevented by the addition of an anti-oxidant molecule, N-acetyl-cysteine. Taken together, our data suggest that Cyclin C, Dnm1 and Ybh3 proteins are important in mediating the TDP-43-induced oxidative stress-mediated cell death in theS. cerevisiaemodel.

It remains controversial whether Dnm1/Drp1-mediated mitochondrial division is essential for mitophagy. Yamashita et al. show that Dnm1/Drp1-independent mitochondrial division occurs after formation of isolation membranes and in cooperation with autophagosome formation during mitophagy., Mitophagy is thought to play an important role in mitochondrial quality control. Mitochondrial division is believed to occur first, and autophagosome formation subsequently occurs to enwrap mitochondria as a process of mitophagy. However, there has not been any temporal analysis of mitochondrial division and autophagosome formation in mitophagy. Therefore, the relationships among these processes remain unclear. We show that the mitochondrial division factor Dnm1 in yeast or Drp1 in mammalian cells is dispensable for mitophagy. Autophagosome formation factors, such as FIP200, ATG14, and WIPIs, were essential for the mitochondrial division for mitophagy. Live-cell imaging showed that isolation membranes formed on the mitochondria. A small portion of the mitochondria then divided from parental mitochondria simultaneously with the extension of isolation membranes and autophagosome formation. These findings suggest the presence of a mitophagy process in which mitochondrial division for mitophagy is accomplished together with autophagosome formation.

Dynamin-related protein 1 (Drp1) is essential for mitochondrial and peroxisomal fission. Recent studies propose that Drp1 does not sever but rather constricts mitochondrial membranes allowing dynamin 2 (Dnm2) to execute final scission. Here, we report that unlike Drp1, Dnm2 is dispensable for peroxisomal and mitochondrial fission, as these events occurred in Dnm2 knockout cells. Fission events were also observed in mouse embryonic fibroblasts lacking Dnm1, 2 and 3. Using reconstitution experiments on preformed membrane tubes, we show that Drp1 alone both constricts and severs membrane tubes. Scission required the membrane binding, self-assembling and GTPase activities of Drp1 and occurred on tubes up to 250 nm in radius. In contrast, Dnm2 exhibited severely restricted fission capacity with occasional severing of tubes below 50 nm in radius. We conclude that Drp1 has both membrane constricting and severing abilities and is the dominant dynamin performing mitochondrial and peroxisomal fission., Drp1 and Dnm2 have been implicated in mitochondrial fission events, although their specific activities in constriction and scission have been unclear. Here, the authors demonstrate that Drp1 is sufficient to constrict and sever mitochondrial and peroxisomal membranes in the absence of Dnm proteins.

Mitochondria and peroxisomes proliferate by division. During division, a part of their membrane is pinched off by constriction of the ring-shaped mitochondrial division (MD) and peroxisome-dividing (POD) machinery. This constriction is mediated by a dynamin-like GTPase Dnm1 that requires a large amount of GTP as an energy source. Here, via proteomics of the isolated division machinery, we show that the 17-kDa nucleoside diphosphate kinase-like protein, dynamin-based ring motive-force organizer 1 (DYNAMO1), locally generates GTP in MD and POD machineries. DYNAMO1 is widely conserved among eukaryotes and colocalizes with Dnm1 on the division machineries. DYNAMO1 converts ATP to GTP, and disruption of its activity impairs mitochondrial and peroxisomal fissions. DYNAMO1 forms a ring-shaped complex with Dnm1 and increases the magnitude of the constricting force. Our results identify DYNAMO1 as an essential component of MD and POD machineries, suggesting that local GTP generation in Dnm1-based machinery regulates motive force for membrane severance., Mitochondria and peroxisomes require a dynamin-like GTPase to remodel membranes during division. Here the authors identify DYNAMO1, a nucleoside diphosphate kinase-like protein that generates a local source of GTP to promote constriction of the division machinery and produce daughter organelles.

Mitochondria fulfill central cellular functions including energy metabolism, iron-sulfur biogenesis, and regulation of apoptosis and calcium homeostasis. Accumulation of dysfunctional mitochondria is observed in ageing and many human diseases such as cancer and various neurodegenerative disorders. Appropriate quality control of mitochondria is important for cell survival in most eukaryotic cells. One important pathway in this respect is mitophagy, a selective form of autophagy which removes excess and dysfunctional mitochondria. In the past decades a series of essential factors for mitophagy have been identified and characterized. However, little is known about the molecular mechanisms regulating mitophagy. The role of mitochondrial dynamics in mitophagy is controversially discussed. Here we will review recent advances in this context promoting our understanding on the molecular regulation of mitophagy in Saccharomyces cerevisiae and on the role of mitochondrial dynamics in mitochondrial quality control. This article is part of a Special Issue entitled: Mitophagy.

Mitochondria are organized as tubular networks in the cell and undergo fission and fusion. While several of the molecular players involved in mediating mitochondrial dynamics have been identified, the precise cellular cues that initiate fission or fusion remain largely unknown. In fission yeast, mitochondria are organized along microtubule bundles. Here, we employed deletions of kinesin-like proteins to perturb microtubule dynamics, and determined that cells with long microtubules exhibited long, but fewer mitochondria, whereas cells with short microtubules exhibited short, but several mitochondria due to reduced mitochondrial fission in the former and elevated fission in the latter. Correspondingly, upon onset of closed mitosis in fission yeast, wherein interphase microtubules assemble to form the spindle within the nucleus, we measured increased mitochondrial fission. We determined that the consequent rise in the mitochondrial copy number was necessary to reduce partitioning errors while stochastically partitioning mitochondria between daughter cells. We discovered that the association of mitochondria with microtubules physically impeded the assembly of the fission protein Dnm1 around mitochondria, resulting in inhibition of mitochondrial fission. Taken together, we demonstrate a novel mechanism for regulation of mitochondrial fission that is dictated by the interaction between mitochondria and the microtubule cytoskeleton.

A novel motif in the Insert B domain of the mitochondrial dynamin Dnm1 is essential for interaction with its adaptor Mdv1 during mitochondrial fission., To initiate mitochondrial fission, dynamin-related proteins (DRPs) must bind specific adaptors on the outer mitochondrial membrane. The structural features underlying this interaction are poorly understood. Using yeast as a model, we show that the Insert B domain of the Dnm1 guanosine triphosphatase (a DRP) contains a novel motif required for association with the mitochondrial adaptor Mdv1. Mutation of this conserved motif specifically disrupted Dnm1–Mdv1 interactions, blocking Dnm1 recruitment and mitochondrial fission. Suppressor mutations in Mdv1 that restored Dnm1–Mdv1 interactions and fission identified potential protein-binding interfaces on the Mdv1 β-propeller domain. These results define the first known function for Insert B in DRP–adaptor interactions. Based on the variability of Insert B sequences and adaptor proteins, we propose that Insert B domains and mitochondrial adaptors have coevolved to meet the unique requirements for mitochondrial fission of different organisms.

Heme is an essential cofactor involved in a plethora of vital functions. Heme is required to be in most subcellular compartments, but the way, in which it is transported from the mitochondria - its site of synthesis - to those compartments is poorly understood. Utilizing ratiometric, fluorescent heme sensors, we found that the GTPases in control of mitochondrial fusion, Mgm1 and fission, Dnm1, are positive and negative regulators of heme trafficking to the nucleus, respectively. Furthermore, we discovered that mitochondrial network morphology is under the control of heme biosynthesis. Our results indicate that mitochondrial dynamics and heme trafficking are integrally coupled.

Yeast peroxisomes multiply by fission. Fission requires two dynamin-related proteins, Dnm1p and Vps1p. Using an in vivo fission assay, we show that Dnm1p-dependent peroxisome fission requires Fis1p, Caf4p and Mdv1p. Fluorescence microscopy of cells expressing GFP-tagged Caf4p and Mdv1p revealed that their association with peroxisomes relies on Fis1p. Vps1p-dependent peroxisome fission occurs independently of these factors. Vps1p contributes most to fission of peroxisomes when cells are grown on glucose. Overexpression of Dnm1p suppresses the fission defect as long as Fis1p and either Mdv1p or Caf4p are present. Conversely, overexpression of Dnm1p does not restore the vacuolar fusion defect of vps1 cells and Vps1p overexpression does not restore the mitochondrial fission defect of dnm1 cells. These data show that Vps1p and Dnm1p are part of independent fission machineries. Because the contribution of Dnm1p to peroxisome fission appears to be more pronounced in cells that proliferate peroxisomes in response to mitochondrial dysfunction, Dnm1p might be part of the mechanism that coordinates mitochondrial and peroxisomal biogenesis.

We show that the dynamin-like proteins Dnm1p and Vps1p are not required for re-introduction of peroxisomes in Hansenula polymorpha pex3 cells upon complementation with PEX3-GFP. Instead, Dnm1p, but not Vps1p, plays a crucial role in organelle proliferation via fission. In H. polymorpha DNM1 deletion cells (dnm1) a single peroxisome is present that forms long extensions, which protrude into developing buds and divide during cytokinesis. Budding pex11.dnm1 double deletion cells lack these peroxisomal extensions, suggesting that the peroxisomal membrane protein Pex11p is required for their formation. Life cell imaging revealed that fluorescent Dnm1p-GFP spots fluctuate between peroxisomes and mitochondria. On the other hand Pex11p is present over the entire organelle surface, but concentrates during fission at the basis of the organelle extension in dnm1 cells. Our data indicate that peroxisome fission is the major pathway for peroxisome multiplication in H. polymorpha. (C) 2007 Elsevier B.V. All rights reserved.

The tail-anchored protein Fis1 is implicated as a passive tether in yeast mitochondrial fission. We probed the functional role of Fis1 Glu-78, whose elevated side chain pKa suggests participation in protein interactions. Fis1 binds partners Mdv1 or Dnm1 tightly, but mutation E78A weakens Fis1 interaction with Mdv1, alters mitochondrial morphology, and abolishes fission in a growth assay. In fis1Δ rescue experiments, Fis1-E78A causes a novel localization pattern in which Dnm1 uniformly coats the mitochondria. By contrast, Fis1-E78A at lower expression levels recruits Dnm1 into mitochondrial punctate structures but fails to support normal fission. Thus, Fis1 makes multiple interactions that support Dnm1 puncta formation and may be essential after this step, supporting a revised model for assembly of the mitochondrial fission machinery. The insights gained by mutating a residue with a perturbed pKa suggest that side chain pKa values inferred from routine NMR sample pH optimization could provide useful leads for functional investigations.

Martin Graef highlights Yamashita et al.’s finding that mitophagy can occur independently of the canonical mitochondrial fission apparatus., Whether or not mitophagy depends on prior mitochondrial fragmentation by the canonical mitochondrial division machinery is controversial. In this issue, Yamashita et al. (2016. J. Cell Biol. https://doi.org/10.1083/jcb.201605093) report that mitochondrial fragments start to bud and divide from mitochondrial tubules when in tight association with forming autophagosomes, but independently of the mitochondrial division factor Drp1/Dnm1.

Summary Yeast mitochondrial division requires the dynamin-related Dnm1 protein. By isolating high-copy suppressors of a dominant-negative Dnm1p mutant, we uncovered an unexpected role in mitochondrial division and inheritance for Num1p, a protein previously shown to facilitate nuclear migration. num1 mutants contain an interconnected network of mitochondrial tubules, remarkably similar to cells lacking Dnm1p, and time-lapse microscopy confirms that mitochondrial fission is greatly reduced in num1Δ cells. We also find that Num1p assembles into punctate structures, which often colocalize with mitochondrial-bound Dnm1p particles. Suggesting a role for both Num1p and Dnm1p in mitochondrial inheritance, we find that num1 dnm1 double mutants accumulate mitochondria in daughter buds and that mother cells are frequently devoid of all mitochondria. Thus, our studies have revealed an additional role for Dnm1p in mitochondrial transmission through its interaction with Num1p, thereby providing a link between mitochondrial division and inheritance.

Mitochondrial DNA (mtDNA) is essential for mitochondrial and cellular function. In Saccharomyces cerevisiae, mtDNA is organized in nucleoprotein structures termed nucleoids, which are distributed throughout the mitochondrial network and are faithfully inherited during the cell cycle. How the cell distributes and inherits mtDNA is incompletely understood although an involvement of mitochondrial fission and fusion has been suggested. We developed a LacO-LacI system to noninvasively image mtDNA dynamics in living cells. Using this system, we found that nucleoids are nonrandomly spaced within the mitochondrial network and observed the spatiotemporal events involved in mtDNA inheritance. Surprisingly, cells deficient in mitochondrial fusion and fission distributed and inherited mtDNA normally, pointing to alternative pathways involved in these processes. We identified such a mechanism, where we observed fission-independent, but F-actin-dependent, tip generation that was linked to the positioning of mtDNA to the newly generated tip. Although mitochondrial fusion and fission were dispensable for mtDNA distribution and inheritance, we show through a combination of genetics and next-generation sequencing that their absence leads to an accumulation of mitochondrial genomes harboring deleterious structural variations that cluster at the origins of mtDNA replication, thus revealing crucial roles for mitochondrial fusion and fission in maintaining the integrity of the mitochondrial genome.

Ageing of biological systems is accompanied by alterations in mitochondrial morphology, including a transformation from networks and filaments to punctuate units. The significance of these alterations with regard to ageing is not known. Here, we demonstrate that the dynamin-related protein 1 (Dnm1p), a mitochondrial fission protein conserved from yeast to humans, affects ageing in the two model systems we studied, Podospora anserina and Saccharomyces cerevisiae. Deletion of the Dnm1 gene delays the transformation of filamentous to punctuate mitochondria and retards ageing without impairing fitness and fertility typically observed in long-lived mutants. Our data further suggest that reduced mitochondrial fission extends life span by increasing cellular resistance to the induction of apoptosis and links mitochondrial dynamics, apoptosis and life-span control.

Yeast mitochondrial fission is a multistep process during which the dynamin-related GTPase, Dnm1p, assembles into punctate structures that associate with the outer mitochondrial membrane and mediate mitochondrial division. Steps in the Dnm1p-dependent process of fission are regulated by the actions of the WD repeat protein, Mdv1p, and the mitochondrial outer membrane protein, Fis1p. Our previous studies suggested a model where Mdv1p functions to regulate fission at a post-Dnm1p assembly step and Fis1p functions at two distinct steps, at an early point, to regulate Dnm1p assembly, and later, together with Mdv1p, to facilitate Dnm1p-dependent mitochondrial fission. To test this model, we have examined the physical and functional relationship between Mdv1p and Fis1p and present genetic, biochemical, and two-hybrid data indicating that a Fis1p–Mdv1p complex is required to regulate mitochondrial fission. To further define the role of Mdv1p in fission, we examined the structural features of Mdv1p required for its interactions with Dnm1p and Fis1p. Data from two-hybrid analyses and GFP-tagged domains of Mdv1p indicate that it contains two functionally distinct domains that enable it to function as a molecular adaptor to regulate sequential interactions between Dnm1p and Fis1p and catalyze a rate-limiting step in mitochondrial fission.

EMBO J 30 14, 2762–2778 (2011); published online June242011 [PMC free article] [PubMed] EMBO Rep 12 6, 565–573 (2011); doi:10.1038/embor.2011.54 [PMC free article] [PubMed] Mitochondria are remarkably dynamic organelles undergoing frequent fusion and fission events. Impairment thereof is linked to numerous neurodegenerative disorders and dysregulation of apoptosis. The principal players mediating mitochondrial fission are considered to be well known and largely conserved between yeast and mammals. However, how the essential fission factor Drp1 is recruited to mitochondria and how its activity is regulated are far more complex than previously assumed. According to a recent study (Otera et al, 2010), recruitment of Drp1 and mitochondrial fission can be exerted by Mff. Surprisingly, these processes do not appear to require Fis1, apparently contradicting several earlier reports on the role of Fis1. Two studies reported in EMBO Reports (Palmer et al, 2011) and in this issue of The EMBO Journal (Zhao et al, 2011) help to shed light on these unexpected findings. They identified two homologous vertebrate-specific negative regulators of Drp1-dependent fission termed: MIEF1/MiD51 and MiD49. They are able to recruit Drp1 to mitochondria but, importantly, rather than promoting fission, bind and inhibit Drp1. In a mutually exclusive manner, MIEF1/MiD51 can form a complex either with Drp1 or with Fis1. Thus, Fis1 may indirectly promote mitochondrial fission by its ability to sequester MIEF1/MiD51, preventing this novel factor from inhibiting mitochondrial fission. Future studies will have to decipher the complex interplay between these novel factors and how they regulate mitochondrial dynamics. In yeast, three factors are known to be required for mitochondrial fission: Dnm1, Fis1 and Mdv1 (Chan, 2006; Westermann, 2010). Of those only Fis1 and Dnm1 have orthologues in vertebrates, namely Fis1 and Drp1, respectively. Drp1, a dynamin-like large GTPase shows a cytosolic localization that after recruitment to mitochondria and oligomerization mediates GTP-dependent fission. Downregulation of Fis1 leads to mitochondrial elongation and overexpression promoted fission consistent with data from yeast (Chan, 2006; Westermann, 2010). Fis1 was proposed to act as the mitochondrial receptor for Drp1-dependent fission (Figure 1). So, it appeared for quite some time in the field that the molecular functions of Drp1 and Fis1 are well conserved from yeast to humans. However, the general role of Fis1 in mitochondrial fission in mammals was questioned recently, for example, with the observation that a conditional knockout of this gene in a carcinoma cell culture model did not lead to a defect in mitochondrial fission, suggesting that Fis1 is dispensable for fission (Otera et al, 2010). The same study analysed in an impressive manner the molecular function of the mitochondrial fission factor, Mff, identified earlier (Gandre-Babbe and van der Bliek, 2008). Otera et al (2010) showed that this factor is able to recruit Drp1 to mitochondria, forms a complex with Drp1 and promotes mitochondrial fission (Figure 1). This was convincingly demonstrated as for example artificially linking Mff to the plasma membrane resulted in Drp1 recruitment to exactly that membrane; and downregulation of Mff led to a reduced number of Drp1-positive foci at the mitochondrial outer membrane accompanied by impairment of mitochondrial fission. In contrast, overexpression of Mff had the opposite effects. These findings strengthened the view that Fis1 is not an essential component of the fission machinery and that Mff may act in addition or alternatively as the mitochondrial receptor for Drp1. The apparent discrepancies to earlier studies in which downregulation of Fis1 impaired fission and upregulation of Fis1 promoted mitochondrial fragmentation were attributed to different types of cell lines and RNAi sequences used (Otera et al, 2010). Figure 1 Models for mitochondrial fission in vertebrates. Classical model, Fis1 acts as the mitochondrial receptor for Drp1 promoting fission. Extended model, Fis1, Mff and/or MIEF1/MiD51/Mid49 recruit Drp1 to the mitochondria. The Mff–Drp1 complex promotes ... However, an additional, attractive hypothesis explaining these findings is now provided by two parallel studies from Zhao et al (2011) and Palmer et al (2011). The latter group identified and characterized the role of MiD49 and MiD51 (mitochondrial dynamics proteins 49/51), which share about 45% amino-acid identity. In parallel, Zhao et al (2011) have identified and characterized the molecular function of MIEF1 (mitochondrial elongation factor 1), which notably is identical to MiD51. These studies demonstrate that MIEF1/MiD51 and MiD49 are able to recruit Drp1 to mitochondria (Figure 1). Importantly, instead of promoting mitochondrial fission, they rather block it by sequestering Drp1. MIEF1/MiD51 was further shown to have a pro-fusion activity independent of Mfn2, a known fusion factor located in the outer membrane. Interestingly, MIEF1/MiD51 is able to form two different protein complexes as it, in an apparently mutually exclusive manner, binds either to Drp1 or to Fis1. Consistently, overexpression of Fis1 partially reduces the inhibitory effect of MIEF1/MiD51 on Drp1. With these findings, these studies provide a rationale how Fis1 may, more indirectly, affect mitochondrial fission and explain part of the apparently contradicting results reported during the last years: Fis1 overexpression would sequester MIEF1/MiD51. That prevents the latter to act as a fission inhibitor as Drp1 cannot be sequestered efficiently anymore. Consequently, mitochondrial fission is promoted. Downregulation of Fis1 would increase the levels of available MIEF1/MiD51 molecules, resulting in a more efficient inactivation of Drp1. This certainly leaves the question why in the study by Otera et al (2010) Fis1 knockdown by various means did not appear to impact mitochondrial fission. We can only speculate, but based on the aforementioned model (Figure 1) lower levels of MIEF1/MiD51 or MiD49 in the cell types analysed could be a possible explanation. Future studies will have to test this. The two studies by Zhao et al (2011) and Palmer et al (2011) are consistent regarding the role of MIEF1/MiD51/MiD49 in recruiting and inhibiting Drp1 function when overexpressed. However, there are some apparent discrepancies when MIEF1/MiD51/MiD49 are downregulated. Depletion of both MIEF1/MiD51 and MiD49 at the same time resulted in mitochondrial elongation and no effect on morphology was observed when only one of these proteins was depleted (Palmer et al, 2011). The authors propose that Drp1 recruitment is hampered when both proteins are depleted, which would explain these observations. They further suggest that MIEF1/MiD51 and MiD49 also can act as a bona fide fission factor as downregulation of MIEF1/MiD51 and MiD49 delayed CCCP-induced mitochondrial fragmentation in a similar way as downregulation of Drp1. In contrast, Zhao et al (2011) observed enhanced mitochondrial fragmentation upon depletion of MIEF1/Mid51. This is in line with the proposed role of MIEF1/Mid51 as a Drp1 inhibitor but requires that recruitment of Drp1 to mitochondria is maintained. Still, as MiD49 was not depleted in this study, and as Fis1 and Mff were still present, this may still occur sufficiently. These few discrepancies between the two studies can for example be attributed to differences in the way of downregulation and the cell lines used. However, we feel that they point to potential differences in the specific roles of MIEF1/MiD51 and MiD49, and it will be interesting to dissect them in the future. Taken together, our view on how mitochondrial fission is regulated and which factors are involved has expanded quite a lot with these studies discussed here. How the different Drp1-containing complexes are formed and whether they can dynamically exchange subunits is an open question. Furthermore, it will be an exciting challenge to reveal the detailed interplay of these factors and how the different complexes are regulated.

Mitochondria are essential organelles found in virtually all eukaryotic cells that play key roles in a variety of cellular processes. Mitochondria show a striking heterogeneity in their number, location, and shape in many different cell types. Although the dynamic nature of mito- chondria has been known for decades, the molecules and mechanisms that mediate these processes are largely unknown. Recently, several laboratories have isolated and analyzed mutants in the yeast Saccharomyces cerevisiae defective in mitochondrial fusion and division, in the segregation of mitochondria to daughter cells, and in the establishment and maintenance of mitochondrial shape. These studies have identified several proteins that appear to mediate different aspects of mito- chondrial morphogenesis. Although it is clear that many additional components have yet to be identified, some of the newly discovered proteins raise intriguing possibilities for how the processes of mitochondrial division, fusion, and segregation occur. Below we summarize our current under- standing of the molecules known to be required for yeast mitochondrial dynamics. Microsc. Res. Tech. 51:573-583, 2000. © 2000 Wiley-Liss, Inc.

Peroxisomes are highly dynamic organelles that have multiple functions in cellular metabolism. To adapt the intracellular conditions to the changing extracellular environment, peroxisomes undergo constitutive segregation and degradation. The segregation of peroxisomes is mediated by 2 dynamin-related GTPases, Dnm1 and Vps1, whereas, the degradation of peroxisomes is accomplished through pexophagy, a selective type of autophagy. During pexophagy, the size of the organelle is always a challenging factor for the efficiency of engulfment by the sequestering compartment, the phagophore, which implies a potential role for peroxisomal fission in the degradation process, similar to the situation with selective mitochondria degradation. In this study, we report that peroxisomal fission is indeed critical for the efficient elimination of the organelle. When pexophagy is induced, both Dnm1 and Vps1 are recruited to the degrading peroxisomes through interactions with Atg11 and Atg36. In addition, we found that specific peroxisomal fission, which is only needed for pexophagy, occurs at mitochondria-peroxisome contact sites.

The Drosophila melanogaster fuzzy onions ( fzo ) gene encodes the first known protein mediator of mitochondrial fusion. During Drosophila spermatogenesis, mitochondria in early postmeiotic spermatids aggregate, fuse, and elongate beside the growing flagellar axoneme. fzo mutant males are defective in this developmentally regulated mitochondrial fusion and are sterile. fzo encodes a large, novel, predicted transmembrane GTPase that becomes detectable on spermatid mitochondria late in meiosis II, just prior to fusion, and disappears soon after fusion is complete. Missense mutations that alter conserved residues required for GTP binding in other GTPases inhibit the fusogenic activity of Fzo in vivo but do not affect its localization. Fzo has homologs of unknown function in mammals, nematodes, and yeast.

The discovery that genetic mutations in several cellular pathways can increase lifespan has lent support to the notion that pharmacological inhibition of aging pathways can be used to extend lifespan and to slow the onset of age-related diseases. However, so far, only few compounds with such activities have been described. Here, we have conducted a chemical genetic screen for compounds that cause the extension of chronological lifespan of Schizosaccharomyces pombe. We have characterized eight natural products with such activities, which has allowed us to uncover so far unknown anti-aging pathways in S. pombe. The ionophores monensin and nigericin extended lifespan by affecting vacuolar acidification, and this effect depended on the presence of the vacuolar ATPase (V-ATPase) subunits Vma1 and Vma3. Furthermore, prostaglandin J2 displayed anti-aging properties due to the inhibition of mitochondrial fission, and its effect on longevity required the mitochondrial fission protein Dnm1 as well as the G-protein-coupled glucose receptor Git3. Also, two compounds that inhibit guanosine monophosphate (GMP) synthesis, mycophenolic acid (MPA) and acivicin, caused lifespan extension, indicating that an imbalance in guanine nucleotide levels impinges upon longevity. We furthermore have identified diindolylmethane (DIM), tschimganine, and the compound mixture mangosteen as inhibiting aging. Taken together, these results reveal unanticipated anti-aging activities for several phytochemicals and open up opportunities for the development of novel anti-aging therapies. © 2013 John Wiley & Sons Ltd and the Anatomical Society.

Dysfunctional mitochondria show a reduced capacity for fusion and, as mitochondrial fission is maintained, become spatially separated from the intact network. By that mechanism, dysfunctional mitochondria have been proposed to be targeted for selective degradation by mitophagy, thereby providing a quality control system for mitochondria. In yeast, conflicting results concerning the role of mitochondrial dynamics in mitophagy have been reported. Here, we investigate the effects on mitophagy of altering mitochondrial fission and fusion, using biochemical, as well as fluorescence-based, assays. Rapamycin-induced mitophagy was shown to depend upon the autophagy-related proteins Atg11, Atg20 and Atg24, confirming that a selective type of autophagy occurred. Both fragmentation of mitochondria and inhibition of oxidative phosphorylation were not sufficient to trigger mitophagy, and neither deletion of the fission factors Dnm1, Fis1, Mdv1 or Caf4 nor expression of dominant-negative variants of Dnm1 impaired mitophagy. The diminished mitophagy initially observed in a Δfis1 mutant was not due to the absence of Fis1 but rather due to a secondary mutation in WHI2, which encodes a factor reported to function in the general stress response and the Ras-protein kinase A (PKA) signaling pathway. We propose that, in yeast, mitochondrial fission is not a prerequisite for the selective degradation of mitochondria, and that mitophagy is linked to the general stress response and the Ras-PKA signaling pathway.

The a2 mating-type-locus gene lga2 of the basidiomycete Ustilago maydis encodes a mitochondrial protein that interferes with mitochondrial morphology and integrity, and that plays a role in uniparental inheritance of mitochondrial DNA. To address the mode of action of Lga2, we investigated its Dnm1 (a dynamin-related protein)-dependent effects. Here, we demonstrate that Dnm1 functions as a mitochondrial fission component in U. maydis and mediates Lga2-induced mitochondrial fragmentation. Mitochondrial fusion occurred very inefficiently in matings of U. maydis wild-type strains, but was strongly stimulated in the absence of dnm1 and highest in either wild-type or Δdnm1 combinations when the a2 partner was deleted in lga2. This indicates that Dnm1 plays a central role in opposing mitochondrial fusion in response to endogenous lga2 expression and that Lga2 additionally inhibits fusion in a dnm1-independent manner. Our results further show that Lga2 does not stimulate increased turnover of the putative fusion protein Fzo1 and causes mitochondrial branching, loss of mitochondrial DNA and fitness reduction independently of dnm1. We conclude that Lga2 acts upstream of Dnm1, but controls mitochondrial integrity independently of Dnm1-mediated fission. In addition, we demonstrate a role of dnm1 in fungal virulence.

Mitochondria are highly dynamic organelles that continuously divide and fuse. These dynamic processes regulate the size, shape, and distribution of the mitochondrial network. In addition, mitochondrial division and fusion play critical roles in cell physiology. This review will focus on the dynamic process of mitochondrial division, which is highly conserved from yeast to humans. We will discuss what is known about how the essential components of the division machinery function to mediate mitochondrial division and then focus on proteins that have been implicated in division but whose functions remain unclear. We will then briefly discuss the cellular functions of mitochondrial division and the problems that arise when division is disrupted.

In mammals, mitochondria are important mediators of programmed cell death, and this process is often regulated by Bcl-2 family proteins. However, a role for mitochondria-mediated cell death in non-mammalian species is more controversial. New evidence from a variety of sources suggests that mammalian mitochondrial fission/division proteins also have the capacity to promote programmed cell death, which may involve interactions with Bcl-2 family proteins. Homologues of these fission factors and several additional mammalian cell death regulators are conserved in flies, worms and yeast, and have been suggested to regulate programmed cell death in these species as well. However, the molecular mechanisms by which these phylogenetically conserved proteins contribute to cell death are not known for any species. Some have taken the conserved pro-death activity of mitochondrial fission factors to mean that mitochondrial fission per se, or failed attempts to undergo fission, are directly involved in cell death. Other evidence suggests that the fission function and the cell death function of these factors are separable. Here we consider the evidence for these arguments and their implications regarding the origins of programmed cell death.

Mitochondria are not produced de novo but are maintained by division. Mitochondrial division is a coordinated process of positioning and constriction of the division site and fission of double membranes, in which dynamin-related protein is believed to mediate outer membrane fission. Part of the mitochondrial division machinery was purified from M phase-arrested Cyanidioschyzon merolae cells through biochemical fractionation. The dynamin-related protein Dnm1 was one of the two major proteins in the purified fraction and was accompanied by a newly identified protein CMR185C, named Mda1. Mda1 contained a predictable coiled-coil region and WD40 repeats, similarly to Mdv1 and Caf4 in yeasts. Immunofluorescence and immunoelectron microscopy showed that Mda1 localizes as a medial belt or ring on the mitochondrial outer surface throughout the division. The ring formation of Mda1 followed the plane of the ring of FtsZ, a protein that resides in the matrix. Dnm1 consistently colocalized with Mda1 only in the late stages of division. Mda1 protein was expressed through S to M phases and was phosphorylated specifically in M phase when Mda1 transformed from belt into foci and became colocalizing with Dnm1. Dephosphorylation of Mda1 in vitro increased its sedimentation coefficient, suggesting conformational changes of the macromolecule. Disassembly of the purified mitochondrial division machinery was performed by adding GTP to independently release Dnm1, suggesting that Mda1 forms a stable homo-oligomer by itself as a core structure of the mitochondrial division machinery.

The mitochondrial division machinery consists of the large dynamin-related protein Dnm1p (Drp1/Dlp1 in humans), and Fis1p, Mdv1p and Caf4p. Proper assembly of Dnm1p complexes on the mitochondrial surface is crucial for balanced fission and fusion events. Using quantitative confocal microscopy, we show that Caf4p is important for the recruitment of Dnm1p to the mitochondria. The mitochondrial Dnm1p assemblies can be divided into at least two morphologically distinguishable fractions. A small subset of these assemblies appear to be present as Dnm1p-spirals (or rings) that encircle tubule constrictions, with seldom more than seven turns. A larger fraction of the Dnm1p assemblies is primarily present at one side of the mitochondrial tubules. We show that a majority of these mitochondria-associated Dnm1p clusters point towards the cell cortex. This polarized orientation is abolished in fis1Δ and caf4Δ yeast cells, but is maintained in mdv1Δ cells and after disruption of the actin cytoskeleton. This study suggests that Caf4p plays a key role in determining the polarized localization of those Dnm1p clusters that are not immediately involved in the mitochondrial fission process.

Mitochondria are the product of an ancient endosymbiotic event between an α‐proteobacterium and an archael host. An early barrier to overcome in this relationship was the control of the bacterium's proliferation within the host. Undoubtedly, the bacterium (or protomitochondrion) would have used its own cell division apparatus to divide at first and, today a remnant of this system remains in some “ancient” and diverse eukaryotes such as algae and amoebae, the most conserved and widespread of all bacterial division proteins, FtsZ. In many of the eukaryotes that still use FtsZ to constrict the mitochondria from the inside, the mitochondria still resemble bacteria in shape and size. Eukaryotes, however, have a mitochondrial morphology that is often highly fluid, and in their tubular networks of mitochondria, division is clearly complemented by mitochondrial fusion. FtsZ is no longer used by these complex eukaryotes, and may have been replaced by other proteins better suited to sustaining complex mitochondrial networks. Although proteins that divide mitochondria from the inside are just beginning to be characterized in higher eukaryotes, many division proteins are known to act on the outside of the organelle. The most widespread of these are the dynamin‐like proteins, which appear to have been recruited very early in the evolution of mitochondria. The essential nature of mitochondria dictates that their loss is intolerable to human cells, and that mutations disrupting mitochondrial division are more likely to be fatal than result in disease. To date, only one disease (Charcot‐Marie‐Tooth disease 2A) has been mapped to a gene that is required for mitochondrial division, whereas two other diseases can be attributed to mutations in mitochondrial fusion genes. Apart from playing a role in regulating the morphology, which might be important for efficient ATP production, research has indicated that the mitochondrial division and fusion proteins can also be important during apoptosis; mitochondrial fragmentation is an early triggering (and under many stimuli, essential) step in the pathway to cell suicide.

Mitochondria form dynamic tubular networks that continually change their shape and move throughout the cell. In eukaryotes, these organellar gymnastics are controlled by numerous pathways that preserve proper mitochondrial morphology and function. The best understood of these are the fusion and fission pathways, which rely on conserved GTPases and their binding partners to regulate organelle connectivity and copy number in healthy cells and during apoptosis. In budding yeast, mitochondrial shape is also maintained by proteins acting in the tubulation pathway. Novel proteins and pathways that control mitochondrial dynamics continue to be discovered, indicating that the mechanisms governing this organelle's behavior are more sophisticated than previously appreciated. Here we review recent advances in the field of mitochondrial dynamics and highlight the importance of these pathways to human health.

Dynamin-related proteins (DRPs) are large self-assembling GTPases whose common function is to regulate membrane dynamics in a variety of cellular processes. Dnm1, which is a yeast DRP (Drp1/Dlp1 in humans), is required for mitochondrial division, but its mechanism is unknown. We provide evidence that Dnm1 likely functions through self-assembly to drive the membrane constriction event that is associated with mitochondrial division. Two regulatory features of Dnm1 self-assembly were also identified. Dnm1 self-assembly proceeded through a rate-limiting nucleation step, and nucleotide hydrolysis by assembled Dnm1 structures was highly cooperative with respect to GTP. Dnm1 formed extended spirals, which possessed diameters greater than those of dynamin-1 spirals but whose sizes, remarkably, were equal to those of mitochondrial constriction sites in vivo. These data suggest that Dnm1 has evolved to form structures that fit the dimensions of mitochondria.

Mitochondria are dynamic organelles that undergo frequent division and fusion, but the molecular mechanisms of these two events are not well understood. Dnm1p, a mitochondria-associated, dynamin-related GTPase was previously shown to mediate mitochondrial fission. Recently, a genome-wide yeast two-hybrid screen identified an uncharacterized protein that interacts with Dnm1p. Cells disrupted in this new gene, which we call NET2, contain a single mitochondrion that consists of a network formed by interconnected tubules, similar to the phenotype of dnm1Δ cells. NET2 encodes a mitochondria-associated protein with a predicted coiled-coil region and six WD-40 repeats. Immunofluorescence microscopy indicates that Net2p is located in distinct, dot-like structures along the mitochondrial surface, many of which colocalize with the Dnm1 protein. Fluorescence and immunoelectron microscopy shows that Dnm1p and Net2p preferentially colocalize at constriction sites along mitochondrial tubules. Our results suggest that Net2p is a new component of the mitochondrial division machinery.

Mitochondria exist as a dynamic tubular network with projections that move, break, and reseal in response to local environmental changes. We present evidence that a human dynamin-related protein (Drp1) is specifically required to establish this morphology. Drp1 is a GTPase with a domain structure similar to that of other dynamin family members. To identify the function of Drp1, we transiently transfected cells with mutant Drp1. A mutation in the GTPase domain caused profound alterations in mitochondrial morphology. The tubular projections normally present in wild-type cells were retracted into large perinuclear aggregates in cells expressing mutant Drp1. The morphology of other organelles was unaffected by mutant Drp1. There was also no effect of mutant Drp1 on the transport functions of the secretory and endocytic pathways. By EM, the mitochondrial aggregates found in cells that were transfected with mutant Drp1 appear as clusters of tubules rather than a large mass of coalescing membrane. We propose that Drp1 is important for distributing mitochondrial tubules throughout the cell. The function of this new dynamin-related protein in organelle morphology represents a novel role for a member of the dynamin family of proteins.

Mitochondrial fission in eukaryotes is mediated by protein complexes that encircle and divide mitochondrial tubules. In budding yeast, fission requires the membrane-anchored protein Fis1 and the dynamin-related GTPase Dnm1. Dnm1 is recruited to mitochondria via interactions with the adaptor proteins Caf4 and Mdv1, which bind directly to Fis1. Unlike Mdv1, a function for Caf4 in mitochondrial membrane scission has not been established. In this study, we demonstrate that Caf4 is a bona fide fission adaptor that assembles at sites of mitochondrial division. We also show that fission complexes may contain Caf4 alone or both Caf4 and Mdv1 without compromising fission function. Although there is a correspondence between Caf4 and Mdv1 expression levels and their contribution to fission, the two adaptor proteins are not equivalent. Rather, our functional and phylogenetic analyses indicate that Caf4 mitochondrial fission activity has diverged from that of Mdv1.

[Figure][1] Wild-type Dnm1 (green, left) localizes to mitochondria (red), but a mutant version (green, right) remains cytosolic, resulting in elongated mitochondria that can’t undergo fission. [Bui et al.][2] investigate how a dynamin-related GTPase is recruited to the mitochondrial

[][1] Dnm1 self-assembles into spirals tailored to fit mitochondria. Dynamin-related proteins regulate membrane dynamics throughout the cell. Ingerman et al. report on [page 1021][2] that these proteins form large polymers that are structurally tailored to fit their individual jobs

Dynamin-related protein 1 (Drp1) belongs to a family of large GTPase proteins that regulate membrane dynamics and morphology. Drp1 localizes to mitochondrial constriction sites in vivo to facilitate outer membrane fission, and mutations that inhibit its activity lead to hyper-fused mitochondria in vivo. Direct inhibition of Drp1 protects against cell death by limiting increased mitochondrial fission associated with apoptosis. Using cryo-electron microscopy (cryo-EM), previous studies of the yeast homolog of Drp1, Dnm1, were used to determine its structural properties. Dnm1 was shown to form large (>100 nm in diameter) helical oligomers that constrict upon GTP hydrolysis to generate a contractile force on the underlying membrane. Similar methods are now being used to gain mechanistic insight into the mammalian mitochondrial fission complex. Several similarities and differences have been found between the yeast and mammalian systems. In solution, Drp1 forms stable tetramers, which represent the pre-assembled state of Drp1. The size of this complex (∼330 kDa) provides a suitable target for 3D image reconstruction. Additional interactions with GTP analogs and/or synthetic liposomes promote Drp1 self-assembly into extended helical oligomers. The 3D structures of these helices will be determined to elucidate interactions that mediate Drp1 self-assembly. The effects of GTP hydrolysis on the Drp1 helical oligomers are also being studied to determine how Drp1 promotes outer mitochondrial membrane fission. Future studies will examine interactions between Drp1 and partner proteins in the mitochondrial fission complex.