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Male germline development involves choreographed changes to mitochondrial number, morphology and organization. Mitochondrial reorganization during spermatogenesis was recently shown to require mitochondrial fusion and fission. Mitophagy, the autophagic degradation of mitochondria, is another mechanism for controlling mitochondrial number and physiology, but its role during spermatogenesis is largely unknown. During post-meiotic spermatid development, restructuring of the mitochondrial network results in packing of mitochondria into a tight array in the sperm midpiece to fuel motility. Here, we show that disruption of mouse Fis1 in the male germline results in early spermatid arrest that is associated with increased mitochondrial content. Mutant spermatids coalesce into multinucleated giant cells that accumulate mitochondria of aberrant ultrastructure and numerous mitophagic and autophagic intermediates, suggesting a defect in mitophagy. We conclude that Fis1 regulates mitochondrial morphology and turnover to promote spermatid maturation., Summary: Analysis of germ cell-specific Fis1 knockout mice and mitophagy reporter mice reveals that the mitochondrial dynamics gene Fis1 regulates mitochondrial morphology and turnover during spermatid maturation.

OPA1, a large GTPase of the dynamin superfamily, mediates fusion of the mitochondrial inner membranes, regulates cristae morphology, and maintains respiratory chain function. Inner membrane–anchored long forms of OPA1 (l-OPA1) are proteolytically processed by the OMA1 or YME1L proteases, acting at cleavage sites S1 and S2, respectively, to produce short forms (s-OPA1). In both mice and humans, half of the mRNA splice forms of Opa1 are constitutively processed to yield exclusively s-OPA1. However, the function of s-OPA1 in mitochondrial fusion has been debated, because in some stress conditions, s-OPA1 is dispensable for fusion. By constructing cells in which the Opa1 locus no longer produces transcripts with S2 cleavage sites, we generated a simplified system to identify the new YME1L-dependent site S3 that mediates constitutive and complete cleavage of OPA1. We show that mitochondrial morphology is highly sensitive to the ratio of l-OPA1 to s-OPA1, indicating that s-OPA1 regulates mitochondrial fusion.

Mitochondria are highly dynamic organelles that continuously grow, divide, and fuse. The division of mitochondria is crucial for human health. During mitochondrial division, the mechano-guanosine triphosphatase (GTPase) dynamin-related protein (Drp1) severs mitochondria at endoplasmic reticulum (ER)-mitochondria contact sites, where peripheral ER tubules interact with mitochondria. Here, we report that Drp1 directly shapes peripheral ER tubules in human and mouse cells. This ER-shaping activity is independent of GTP hydrolysis and located in a highly conserved peptide of 18 amino acids (termed D-octadecapeptide), which is predicted to form an amphipathic α helix. Synthetic D-octadecapeptide tubulates liposomes in vitro and the ER in cells. ER tubules formed by Drp1 promote mitochondrial division by facilitating ER-mitochondria interactions. Thus, Drp1 functions as a two-in-one protein during mitochondrial division, with ER tubulation and mechano-GTPase activities.

After birth, cardiomyocytes (CM) acquire numerous adaptations in order to efficiently pump blood throughout an animal’s lifespan. How this maturation process is regulated and coordinated is poorly understood. Here, we perform a CRISPR/Cas9 screen in mice and identify serum response factor (SRF) as a key regulator of CM maturation. Mosaic SRF depletion in neonatal CMs disrupts many aspects of their maturation, including sarcomere expansion, mitochondrial biogenesis, transverse-tubule formation, and cellular hypertrophy. Maintenance of maturity in adult CMs is less dependent on SRF. This stage-specific activity is associated with developmentally regulated SRF chromatin occupancy and transcriptional regulation. SRF directly activates genes that regulate sarcomere assembly and mitochondrial dynamics. Perturbation of sarcomere assembly but not mitochondrial dynamics recapitulates SRF knockout phenotypes. SRF overexpression also perturbs CM maturation. Together, these data indicate that carefully balanced SRF activity is essential to promote CM maturation through a hierarchy of cellular processes orchestrated by sarcomere assembly.

Differentiating cells tailor their metabolism to fulfill their specialized functions. We examined whether mitochondrial fusion is important for metabolic tailoring during spermatogenesis. Acutely after depletion of mitofusins Mfn1 and Mfn2, spermatogenesis arrests due to failure to accomplish a metabolic shift during meiosis. This metabolic shift includes increased mitochondrial content, mitochondrial elongation, and upregulation of oxidative phosphorylation (OXPHOS). With long-term mitofusin loss, all differentiating germ cell types are depleted, but proliferation of stem-like undifferentiated spermatogonia remains unaffected. Thus, compared with undifferentiated spermatogonia, differentiating spermatogonia and meiotic spermatocytes have cell physiologies that require high levels of mitochondrial fusion. Proteomics in fibroblasts reveals that mitofusin-null cells downregulate respiratory chain complexes and mitochondrial ribosomal subunits. Similarly, mitofusin depletion in immortalized spermatocytes or germ cells in vivo results in reduced OXPHOS subunits and activity. We reveal that by promoting OXPHOS, mitofusins enable spermatogonial differentiation and a metabolic shift during meiosis.

DNM1L encodes dynamin-related protein 1 (DRP1/DLP1), a key component of the mitochondrial fission machinery that is essential for proper functioning of the mammalian brain. Previously reported probands with de novo missense mutations in DNM1L presented in the first year of life with severe encephalopathy and refractory epilepsy, with several dying within the first several weeks after birth. In contrast, we report identical novel missense mutations in DNM1L in two unrelated probands who experienced normal development for several years before presenting with refractory focal status epilepticus and subsequent rapid neurological decline. We expand the phenotype of DNM1L-related mitochondrial fission defects, reveal common unique clinical characteristics and imaging findings, and compare the cellular impact of this novel mutation to the previously reported A395D lethal variant. We demonstrate that our R403C mutation, which resides in the assembly region of DRP1, acts by a dominant-negative mechanism and reduces oligomerization, mitochondrial fission activity, and mitochondrial recruitment of DRP1, but to a lesser extent compared to the A395D mutation. In contrast to the initial report of neonatal lethality resulting from DNM1L mutation and DRP1 dysfunction, our results show that milder DRP1 impairment is compatible with normal early development and subsequently results in a distinct set of neurological findings. In addition, we identify a common pathogenic mechanism whereby DNM1L mutations impair mitochondrial fission. © 2016 Wiley Periodicals, Inc.

OPA1 is the major gene responsible for Dominant Optic Atrophy (DOA) and the syndromic form DOA “plus”. Over 370 OPA1 mutations have been identified so far, although their pathogenicity is not always clear. We have analyzed one novel and a set of known OPA1 mutations to investigate their impact on protein functions in primary skin fibroblasts and in two “ad hoc” generated cell systems: the MGM1/OPA1 chimera yeast model and the Opa1−/− MEFs model expressing the mutated human OPA1 isoform 1. The yeast model allowed us to confirm the deleterious effects of these mutations and to gain information on their dominance/recessivity. The MEFs model enhanced the phenotypic alteration caused by mutations, nicely correlating with the clinical severity observed in patients, and suggested that the DOA “plus” phenotype could be induced by the combinatorial effect of mitochondrial network fragmentation with variable degrees of mtDNA depletion. Overall, the two models proved to be valuable tools to functionally assess and define the deleterious mechanism and the pathogenicity of novel OPA1 mutations, and useful to testing new therapeutic interventions.

Summary: OPA1 is a GTPase that controls mitochondrial fusion, cristae integrity, and mtDNA maintenance. In humans, eight isoforms are expressed as combinations of long and short forms, but it is unclear whether OPA1 functions are associated with specific isoforms and/or domains. To address this, we expressed each of the eight isoforms or different constructs of isoform 1 in Opa1−/− MEFs. We observed that any isoform could restore cristae structure, mtDNA abundance, and energetic efficiency independently of mitochondrial network morphology. Long forms supported mitochondrial fusion; short forms were better able to restore energetic efficiency. The complete rescue of mitochondrial network morphology required a balance of long and short forms of at least two isoforms, as shown by combinatorial isoform silencing and co-expression experiments. Thus, multiple OPA1 isoforms are required for mitochondrial dynamics, while any single isoform can support all other functions. These findings will be useful in designing gene therapies for patients with OPA1 haploinsufficiency. : Del Dotto et al. perform a systematic analysis of the function of each of the eight OPA1 isoforms. They find that any OPA1 isoform can rescue mtDNA content, cristae structure, and mitochondrial energetics. A specific combination of long and short forms is required for mitochondrial dynamics and network morphology. Keywords: dominant optic atrophy, mitochondrial network dynamics, mtDNA, OPA1 isoforms, OPA1 long-short form balance

Because the deubiquitinating enzyme USP33 is involved in several important cellular processes (β-adrenergic receptor recycling, centrosome amplification, RalB signaling, and cancer cell migration), its levels must be carefully regulated. Using quantitative mass spectrometry, we found that the intracellular level of USP33 is highly sensitive to the activity of p97. Knockdown or chemical inhibition of p97 causes robust accumulation of USP33 due to inhibition of its degradation. The p97 adaptor complex involved in this function is the Ufd1-Npl4 heterodimer. Furthermore, we identified HERC2, a HECT domain-containing E3 ligase, as being responsible for polyubiquitination of USP33. Inhibition of p97 causes accumulation of polyubiquitinated USP33, suggesting that p97 is required for postubiquitination processing. Thus, our study has identified several key molecules that control USP33 degradation within the ubiquitin-proteasome system.

A defining feature of mitochondria is their maternal mode of inheritance. However, little is understood about the cellular mechanism through which paternal mitochondria, delivered from sperm, are eliminated from early mammalian embryos. Autophagy has been implicated in nematodes, but whether this mechanism is conserved in mammals has been disputed. Here, we show that cultured mouse fibroblasts and pre-implantation embryos use a common pathway for elimination of mitochondria. Both situations utilize mitophagy, in which mitochondria are sequestered by autophagosomes and delivered to lysosomes for degradation. The E3 ubiquitin ligases PARKIN and MUL1 play redundant roles in elimination of paternal mitochondria. The process is associated with depolarization of paternal mitochondria and additionally requires the mitochondrial outer membrane protein FIS1, the autophagy adaptor P62, and PINK1 kinase. Our results indicate that strict maternal transmission of mitochondria relies on mitophagy and uncover a collaboration between MUL1 and PARKIN in this process. DOI: http://dx.doi.org/10.7554/eLife.17896.001, eLife digest Mitochondria are commonly referred to as the 'powerhouses' of animal cells because these structures provide the majority of the energy in most cells. People inherit their mitochondria from their mother, and not their father. This is because the father's mitochondria, which are delivered by sperm to the egg, are degraded early on when the embryo starts to develop. Previous studies with model organisms, like nematode worms, showed that mitochondria delivered via sperm (also known as 'paternal mitochondria') were delivered to structures called lysosomes and broken down by the enzymes contained within. However, it remained controversial whether this process, named mitophagy, also occurred in mammalian cells, and the molecules involved were unknown. Now, Rojansky et al. have identified key molecules that are essential for the degradation of mitochondria in mouse cells and show that these same molecules are needed to degrade paternal mitochondria in early mouse embryos. These results indicate that paternal mitochondria are indeed degraded by mitophagy in mice. In addition, Rojansky et al. also note that one of the key molecules is a protein called PARKIN, which is mutated in many inherited cases of Parkinson's disease, a major neurodegenerative disorder. Even though these new findings provide a clearer idea as to how paternal mitochondria are degraded, the question of why remains unanswered. As a result, it is likely that this topic will continue to be heavily debated. Nevertheless, having identified the key molecules involved in degrading paternal mitochondria, it may now be possible to address this question more directly – for example by interfering with this process and then examining the consequences. DOI: http://dx.doi.org/10.7554/eLife.17896.002

Mitochondrial dynamics during nutrient starvation of cancer cells likely exert profound effects on their capability for metastatic progression. Here, we report that KAP1 (TRIM28), a transcriptional coadaptor protein implicated in metastatic progression in breast cancer, is a pivotal regulator of mitochondrial fusion in glucose-starved cancer cells. Diverse metabolic stresses induced Ser473 phosphorylation of KAP1 (pS473-KAP1) in a ROS- and p38-dependent manner. Results from live-cell imaging and molecular studies revealed that during the first 6 to 8 hours of glucose starvation, mitochondria initially underwent extensive fusion, but then subsequently fragmented in a pS473-KAP1-dependent manner. Mechanistic investigations using phosphorylation-defective mutants revealed that KAP1 Ser473 phosphorylation limited mitochondrial hyperfusion in glucose-starved breast cancer cells, as driven by downregulation of the mitofusin protein MFN2, leading to reduced oxidative phosphorylation and ROS production. In clinical specimens of breast cancer, reduced expression of MFN2 corresponded to poor prognosis in patients. In a mouse xenograft model of human breast cancer, there was an association in the core region of tumors between MFN2 downregulation and the presence of highly fragmented mitochondria. Collectively, our results suggest that KAP1 Ser473 phosphorylation acts through MFN2 reduction to restrict mitochondrial hyperfusion, thereby contributing to cancer cell survival under conditions of sustained metabolic stress. Cancer Res; 76(17); 5006–18. ©2016 AACR.

Mice deficient for mitochondrial fission factor (Mff) die from severe cardiomyopathy, but cardiac function, tissue integrity, and mitochondrial respiration are robustly rescued by rebalancing mitochondrial dynamics through deletion of Mitofusin 1 (Mfn1)., Defects in mitochondrial fusion or fission are associated with many pathologies, raising the hope that pharmacological manipulation of mitochondrial dynamics may have therapeutic benefit. This approach assumes that organ physiology can be restored by rebalancing mitochondrial dynamics, but this concept remains to be validated. We addressed this issue by analyzing mice deficient in Mff, a protein important for mitochondrial fission. Mff mutant mice die at 13 wk as a result of severe dilated cardiomyopathy leading to heart failure. Mutant tissue showed reduced mitochondrial density and respiratory chain activity along with increased mitophagy. Remarkably, concomitant deletion of the mitochondrial fusion gene Mfn1 completely rescued heart dysfunction, life span, and respiratory chain function. Our results show for the first time that retuning the balance of mitochondrial fusion and fission can restore tissue integrity and mitochondrial physiology at the whole-organ level. Examination of liver, testis, and cerebellum suggest, however, that the precise balance point of fusion and fission is cell type specific.

Parkin, an E3 ubiquitin ligase implicated in Parkinson's disease, promotes degradation of dysfunctional mitochondria by autophagy. Using proteomic and cellular approaches, we show that upon translocation to mitochondria, Parkin activates the ubiquitin–proteasome system (UPS) for widespread degradation of outer membrane proteins. This is evidenced by an increase in K48-linked polyubiquitin on mitochondria, recruitment of the 26S proteasome and rapid degradation of multiple outer membrane proteins. The degradation of proteins by the UPS occurs independently of the autophagy pathway, and inhibition of the 26S proteasome completely abrogates Parkin-mediated mitophagy in HeLa, SH-SY5Y and mouse cells. Although the mitofusins Mfn1 and Mfn2 are rapid degradation targets of Parkin, we find that degradation of additional targets is essential for mitophagy. These results indicate that remodeling of the mitochondrial outer membrane proteome is important for mitophagy, and reveal a causal link between the UPS and autophagy, the major pathways for degradation of intracellular substrates.

The dynamin-related GTPase OPA1 is mutated in autosomal dominant optic atrophy (DOA) (Kjer type), an inherited neuropathy of the retinal ganglion cells. OPA1 is essential for the fusion of the inner mitochondrial membranes, but its mechanism of action remains poorly understood. Here we show that OPA1 has a low basal rate of GTP hydrolysis that is dramatically enhanced by association with liposomes containing negative phospholipids such as cardiolipin. Lipid association triggers assembly of OPA1 into higher order oligomers. In addition, we find that OPA1 can promote the protrusion of lipid tubules from the surface of cardiolipin-containing liposomes. In such lipid protrusions, OPA1 assemblies are observed on the outside of the lipid tubule surface, a protein-membrane topology similar to that of classical dynamins. The membrane tubulation activity of OPA1 is suppressed by GTPgammaS. OPA1 disease alleles associated with DOA display selective defects in several activities, including cardiolipin association, GTP hydrolysis and membrane tubulation. These findings indicate that interaction of OPA1 with membranes can stimulate higher order assembly, enhance GTP hydrolysis and lead to membrane deformation into tubules.

SummaryMutations in the mitochondrial fusion gene Mfn2 cause the human neurodegenerative disease Charcot-Marie-Tooth type 2A. However, the cellular basis underlying this relationship is poorly understood. By removing Mfn2 from the cerebellum, we established a model for neurodegeneration caused by loss of mitochondrial fusion. During development and after maturity, Purkinje cells require Mfn2 but not Mfn1 for dendritic outgrowth, spine formation, and cell survival. In vivo, cell culture, and electron microscopy studies indicate that mutant Purkinje cells have aberrant mitochondrial distribution, ultrastructure, and electron transport chain activity. In fibroblasts lacking mitochondrial fusion, the majority of mitochondria lack mitochondrial DNA nucleoids. This deficiency provides a molecular mechanism for the dependence of respiratory activity on mitochondrial fusion. Our results show that exchange of mitochondrial contents is important for mitochondrial function as well as organelle distribution in neurons and have important implications for understanding the mechanisms of neurodegeneration due to perturbations in mitochondrial fusion.

Mitochondria are mobile organelles that dynamically remodel their membranes and actively migrate along cytoskeletal tracks. There is overwhelming evidence that regulators of mitochondrial dynamics are critical for the survival and function of neural tissues. In multiple animal models, ablation of genes regulating mitochondrial shape result in stunted neural development and neurodegeneration. Organotypic cultures serve as ideal in vitro tissue models to further dissect the mechanisms of mitochondrial function in neuronal survival. Slice cultures preserve the three-dimensional cytoarchitecture of neural networks and can survive for prolonged periods in culture. In addition, these cultures allow long-term assessment of genetic or pharmacologic perturbations on neuronal function. Organotypic preparations from murine and rat models have been developed for many regions of the brain. In this chapter, we describe our methods for preparing basal ganglia and cerebellar slice cultures suitable for studying mitochondrial function in Parkinson's disease and cerebellar ataxia, respectively. With such slices, we describe a robust method for live imaging of mitochondrial dynamics. To quantitatively analyze mitochondrial motility, we show how to generate kymographs using the open source image analysis program ImageJ. These techniques provide a powerful platform for assessing mitochondrial activity in neural networks.

Mitochondria provide a myriad of services to the cell, including energy production, calcium buffering and regulation of apoptosis. How these diverse functions are coordinated among the hundreds of mitochondria in a given cell is largely unknown, but is probably dependent on the dynamic nature of mitochondria. In this review, we explore the latest developments in mitochondrial dynamics in mammals. These studies indicate that mitofusins and OPA1 are essential for mitochondrial fusion, whereas Fis1 and Drp1 are essential for mitochondrial fission. The overall morphology of the mitochondrial population depends on the relative activities of these two sets of proteins. In addition to the regulation of mitochondrial shape, these molecules also play important roles in cell and tissue physiology. Perturbation of mitochondrial fusion results in defects in mitochondrial membrane potential and respiration, poor cell growth and increased susceptibility to cell death. These cellular observations may explain why mitochondrial fusion is essential for embryonic development. Two inherited neuropathies, Charcot-Marie-Tooth type 2A and autosomal dominant optic atrophy, are caused by mutations in mitofusin 2 and OPA1, suggesting that proper regulation of mitochondrial dynamics is particularly vital to neurons. Mitochondrial fission accompanies several types of apoptotic cell death and appears important for progression of the apoptotic pathway. These studies provide insight into how mitochondria communicate with one another to coordinate mitochondrial function and morphology.

Mitochondria undergo continual cycles of fusion and fission, and the balance of these opposing processes regulates mitochondrial morphology. Paradoxically, cells invest many resources to maintain tubular mitochondrial morphology, when reducing both fusion and fission simultaneously achieves the same end. This observation suggests a requirement for mitochondrial fusion, beyond maintenance of organelle morphology. Here, we show that cells with targeted null mutations in Mfn1 or Mfn2 retained low levels of mitochondrial fusion and escaped major cellular dysfunction. Analysis of these mutant cells showed that both homotypic and heterotypic interactions of Mfns are capable of fusion. In contrast, cells lacking both Mfn1 and Mfn2 completely lacked mitochondrial fusion and showed severe cellular defects, including poor cell growth, widespread heterogeneity of mitochondrial membrane potential, and decreased cellular respiration. Disruption of OPA1 by RNAi also blocked all mitochondrial fusion and resulted in similar cellular defects. These defects in Mfn-null or OPA1-RNAi mammalian cells were corrected upon restoration of mitochondrial fusion, unlike the irreversible defects found in fzodelta yeast. In contrast, fragmentation of mitochondria, without severe loss of fusion, did not result in such cellular defects. Our results showed that key cellular functions decline as mitochondrial fusion is progressively abrogated.

Infection by human immunodeficiency virus type I requires the fusogenic activity of gp41, the transmembrane subunit of the viral envelope protein. Crystallographic studies have revealed that fusion-active gp41 is a "trimer-of-hairpins" in which three central N-terminal helices form a trimeric coiled coil surrounded by three antiparallel C-terminal helices. This structure is stabilized primarily by hydrophobic, interhelical interactions, and several critical contacts are made between residues that form a deep cavity in the N-terminal trimer and the C-helix residues that pack into this cavity. In addition, the trimer-of-hairpins structure has an extensive network of hydrogen bonds within a conserved glutamine-rich layer of poorly understood function. Formation of the trimer-of-hairpins structure is thought to directly force the viral and target membranes together, resulting in membrane fusion and viral entry. We test this hypothesis by constructing four series of gp41 mutants with disrupted interactions between the N- and C-helices. Notably, in the three series containing mutations within the cavity, gp41 activity correlates well with the stability of the N-C interhelical interaction. In contrast, a fourth series of mutants involving the glutamine layer residue Gln-653 show fusion defects even though the stability of the hairpin is close to wild-type. These results provide evidence that gp41 hairpin stability is critical for mediating fusion and suggest a novel role for the glutamine layer in gp41 function.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein is composed of a complex between the surface subunit gp120, which binds to cellular receptors, and the transmembrane subunit gp41. Upon activation of the envelope glycoprotein by cellular receptors, gp41 undergoes conformational changes that mediate fusion of the viral and cellular membranes. Prior to formation of a fusogenic "trimer-of-hairpins" structure, gp41 transiently adopts a prefusogenic conformation whose structural features are poorly understood. An important approach toward understanding structural conformations of gp41 during HIV-1 entry has been to analyze the structural targets of gp41 inhibitors. We have constructed epitope-tagged versions of 5-Helix, a designed protein that binds to the C-peptide region of gp41 and inhibits HIV-1 membrane fusion. Using these 5-Helix variants, we examined which conformation of gp41 is the target of 5-Helix. We find that although 5-Helix binds poorly to native gp41, it binds strongly to gp41 activated by interaction of the envelope protein with either soluble CD4 or membrane-bound cellular receptors. This preferential interaction with activated gp41 results in the accumulation of 5-Helix on the surface of activated cells. These results strongly suggest that the gp41 prefusogenic intermediate is the target of 5-Helix and that this intermediate has a remarkably "open" structure, with exposed C-peptide regions. These results provide important structural information about this intermediate that should facilitate the development of HIV-1 entry inhibitors and may lead to new vaccine strategies.

Mitochondrial morphology is determined by a dynamic equilibrium between organelle fusion and fission, but the significance of these processes in vertebrates is unknown. The mitofusins, Mfn1 and Mfn2, have been shown to affect mitochondrial morphology when overexpressed. We find that mice deficient in either Mfn1 or Mfn2 die in midgestation. However, whereas Mfn2 mutant embryos have a specific and severe disruption of the placental trophoblast giant cell layer, Mfn1-deficient giant cells are normal. Embryonic fibroblasts lacking Mfn1 or Mfn2 display distinct types of fragmented mitochondria, a phenotype we determine to be due to a severe reduction in mitochondrial fusion. Moreover, we find that Mfn1 and Mfn2 form homotypic and heterotypic complexes and show, by rescue of mutant cells, that the homotypic complexes are functional for fusion. We conclude that Mfn1 and Mfn2 have both redundant and distinct functions and act in three separate molecular complexes to promote mitochondrial fusion. Strikingly, a subset of mitochondria in mutant cells lose membrane potential. Therefore, mitochondrial fusion is essential for embryonic development, and by enabling cooperation between mitochondria, has protective effects on the mitochondrial population.

Mitochondrial morphology is regulated by the balance between two counteracting mitochondrial processes of fusion and fission. There is significant evidence suggesting a stringent association between morphology and bioenergetics of mitochondria. Morphological alterations in mitochondria are linked to several pathological disorders, including cardiovascular diseases. The consequences of stress-induced acetylation of mitochondrial proteins on the organelle morphology remain largely unexplored. Here we report that OPA1, a mitochondrial fusion protein, was hyperacetylated in hearts under pathological stress and this posttranslational modification reduced the GTPase activity of the protein. The mitochondrial deacetylase SIRT3 was capable of deacetylating OPA1 and elevating its GTPase activity. Mass spectrometry and mutagenesis analyses indicated that in SIRT3-deficient cells OPA1 was acetylated at lysine 926 and 931 residues. Overexpression of a deacetylation-mimetic version of OPA1 recovered the mitochondrial functions of OPA1-null cells, thus demonstrating the functional significance of K926/931 acetylation in regulating OPA1 activity. Moreover, SIRT3-dependent activation of OPA1 contributed to the preservation of mitochondrial networking and protection of cardiomyocytes from doxorubicin-mediated cell death. In summary, these data indicated that SIRT3 promotes mitochondrial function not only by regulating activity of metabolic enzymes, as previously reported, but also by regulating mitochondrial dynamics by targeting OPA1.

SummaryMitochondrial fusion is essential for maintenance of mitochondrial function. The mitofusin GTPases control mitochondrial outer membrane fusion, whereas the dynamin-related GTPase Opa1 mediates inner membrane fusion. We show that mitochondrial inner membrane fusion is tuned by the level of oxidative phosphorylation (OXPHOS), whereas outer membrane fusion is insensitive. Consequently, cells from patients with pathogenic mtDNA mutations show a selective defect in mitochondrial inner membrane fusion. In elucidating the molecular mechanism of OXPHOS-stimulated fusion, we uncover that real-time proteolytic processing of Opa1 stimulates mitochondrial inner membrane fusion. OXPHOS-stimulated mitochondrial fusion operates through Yme1L, which cleaves Opa1 more efficiently under high OXPHOS conditions. Engineered cleavage of Opa1 is sufficient to mediate inner membrane fusion, regardless of respiratory state. Proteolytic cleavage therefore stimulates the membrane fusion activity of Opa1, and this feature is exploited to dynamically couple mitochondrial fusion to cellular metabolism.

Mitochondrial fission requires recruitment of the GTPase Drp1 to mitochondria, but the molecules that mediate this recruitment have been disputed. Fis1, Mff, MiD49, and MiD51 can all recruit Drp1 to mitochondria and promote fission. MiD49 and MiD51 can promote mitochondrial fission, but their activity depends on cellular context., Several mitochondrial outer membrane proteins—mitochondrial fission protein 1 (Fis1), mitochondrial fission factor (Mff), mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51, respectively)—have been proposed to promote mitochondrial fission by recruiting the GTPase dynamin-related protein 1 (Drp1), but fundamental issues remain concerning their function. A recent study supported such a role for Mff but not for Fis1. In addition, it is unclear whether MiD49 and MiD51 activate or inhibit fission, because their overexpression causes extensive mitochondrial elongation. It is also unknown whether these proteins can act in the absence of one another to mediate fission. Using Fis1-null, Mff-null, and Fis1/Mff-null cells, we show that both Fis1 and Mff have roles in mitochondrial fission. Moreover, immunofluorescence analysis of Drp1 suggests that Fis1 and Mff are important for the number and size of Drp1 puncta on mitochondria. Finally, we find that either MiD49 or MiD51 can mediate Drp1 recruitment and mitochondrial fission in the absence of Fis1 and Mff. These results demonstrate that multiple receptors can recruit Drp1 to mediate mitochondrial fission.

The murine limb deformity (ld) locus encodes a set of proteins, termed formins, that are required for embryonic limb and kidney development. Previous studies had indicated that these proteins are located in the nucleus and cytoplasm and have biochemical properties consistent with an action within the nucleus. To test the notion that nuclear localization is crucial for formin function, we carried out molecular and biochemical studies on three ld alleles. We show that two transgene-induced alleles, ldTgHd and ldTgBri, generate similar COOH-truncated formins that lack the terminal 110 amino acids, while a third allele, ldIn2, generates a less extensively truncated formin that lacks the terminal 42 amino acids. Using subcellular fractionation analysis, we find that wild-type formin is detected in both nuclear and cytosolic fractions; in contrast, the truncated formins encoded by ldTgHd and ldTgBri are strictly cytosolic. The less extensively truncated ldIn2 formin shows a similar, but less complete, localization defect. Consistent with this weaker cellular phenotype, hind limbs from ldIn2 mice have milder skeletal defects than those of ldTgBri mice. These observations define a small region in the carboxyl terminus that is required for nuclear localization and suggest that nuclear localization plays a role in formin action.

Mice homozygous for the recessive limb deformity (ld) mutation display both limb and renal defects. The limb defects, oligodactyly and syndactyly, have been traced to improper differentiation of the apical ectodermal ridge (AER) and shortening of the anteroposterior limb axis. The renal defects, usually aplasia, are thought to result from failure of ureteric bud outgrowth. Since the ld locus gives rise to multiple RNA isoforms encoding several different proteins (termed formins), we wished to understand their role in the formation of these organs. Therefore, we first examined the embryonic expression patterns of the four major ld mRNA isoforms. Isoforms I, II and III (all con-taining a basic amino terminus) are expressed in dorsal root ganglia, cranial ganglia and the developing kidney including the ureteric bud. Isoform IV (containing an acidic amino terminus) is expressed in the notochord, the somites, the apical ectodermal ridge (AER) of the limb bud and the developing kidney including the ureteric bud. Using a lacZ reporter assay in transgenic mice, we show that this differential expression of isoform IV results from distinct regulatory sequences upstream of its first exon. These expression patterns suggest that all four isoforms may be involved in ureteric bud outgrowth, while isoform IV may be involved in AER differentiation. To define further the developmental consequences of the ld limb defect, we analyzed the expression of a number of genes thought to play a role in limb development. Most signifi-cantly, we find that although the AERs of ld limb buds express several AER markers, they do not express detectable levels of fibroblast growth factor 4 (fgf-4), which has been proposed to be the AER signal to the mesoderm. Thus we conclude that one or more formins are necessary to initiate and/or maintain fgf-4 production in the distal limb. Since ld limbs form distal structures such as digits, we further conclude that while fgf-4 is capable of support-ing distal limb outgrowth in manipulated limbs, it is not essential for distal outgrowth in normal limb development. In addition, ld limbs show a severe decrease in the expression of several mesodermal markers, including sonic hedgehog (shh), a marker for the polarizing region and Hoxd-12, a marker for posterior mesoderm. We propose that incomplete differentiation of the AER in ld limb buds leads to reduction of polarizing activity and defects along the anteroposterior axis.

Mitochondria are dynamic organelles that continually undergo fusion and fission. These opposing processes work in concert to maintain the shape, size, and number of mitochondria and their physiological function. Some of the major molecules mediating mitochondrial fusion and fission in mammals have been discovered, but the underlying molecular mechanisms are only partially unraveled. In particular, the cast of characters involved in mitochondrial fission needs to be clarified. By enabling content mixing between mitochondria, fusion and fission serve to maintain a homogeneous and healthy mitochondrial population. Mitochondrial dynamics has been linked to multiple mitochondrial functions, including mitochondrial DNA stability, respiratory capacity, apoptosis, response to cellular stress, and mitophagy. Because of these important functions, mitochondrial fusion and fission are essential in mammals, and even mild defects in mitochondrial dynamics are associated with disease. A better understanding of these processes likely will ultimately lead to improvements in human health.

Despite growing interest and a recent surge in papers, the role of autophagy in glucose and lipid metabolism is unclear. We produced mice with skeletal muscle–specific deletion of Atg7 (encoding autophagy-related 7). Unexpectedly, these mice showed decreased fat mass and were protected from diet-induced obesity and insulin resistance; this phenotype was accompanied by increased fatty acid oxidation and browning of white adipose tissue (WAT) owing to induction of fibroblast growth factor 21 (Fgf21). Mitochondrial dysfunction induced by autophagy deficiency increased Fgf21 expression through induction of Atf4, a master regulator of the integrated stress response. Mitochondrial respiratory chain inhibitors also induced Fgf21 in an Atf4-dependent manner. We also observed induction of Fgf21, resistance to diet-induced obesity and amelioration of insulin resistance in mice with autophagy deficiency in the liver, another insulin target tissue. These findings suggest that autophagy deficiency and subsequent mitochondrial dysfunction promote Fgf21 expression, a hormone we consequently term a 'mitokine', and together these processes promote protection from diet-induced obesity and insulin resistance.

Many pathological states involve dysregulation of mitochondrial fusion, fission, or transport. These dynamic events are usually studied in cells lines because of the challenges in tracking mitochondria in tissues. To investigate mitochondrial dynamics in tissues and disease models, we generated two mouse lines withphoto-activatable mitochondria (PhAM). In the PhAM ^(floxed) line, a mitochondrially localized version of the photo-convertible fluorescent protein Dendra2 (mito-Dendra2) is targeted to the ubiquitously expressed Rosa26 locus, along with an upstream loxP-flanked termination signal. Expression of Cre in PhAM ^(floxed) cells results in bright mito-Dendra2 fluorescence without adverse effects on mitochondrial morphology. When crossed with Cre drivers, the PhAM ^(floxed) line expresses mito-Dendra2 in specific cell types, allowing mitochondria to be tracked even in tissues that have high cell density. In a second line (PhAM ^(excised)), the expression of mito-Dendra2 is ubiquitous, allowing mitochondria to be analyzed in a wide range of live and fixed tissues. By using photo-conversion techniques, we directly measured mitochondrial fusion events in cultured cells as well as tissues such as skeletal muscle. These mouse lines facilitate analysis of mitochondrial dynamics in a wide spectrum of primary cells and tissues, and can be used to examine mitochondria in developmental transitions and disease states.

Oxidative stress causes mitochondrial fragmentation and dysfunction in age-related diseases through unknown mechanisms. Cardiolipin (CL) is a phospholipid required for mitochondrial oxidative phosphorylation. The function of CL is determined by its acyl composition, which is significantly altered by the onset of age-related diseases. Here, we examine a role of acyl-CoA:lysocardiolipin acyltransferase lysocardiolipin acyltransferase 1 (ALCAT1), a lysocardiolipin acyltransferase that catalyzes pathological CL remodeling, in mitochondrial biogenesis. We show that overexpression of ALCAT1 causes mitochondrial fragmentation through oxidative stress and depletion of mitofusin mitofusin 2 (MFN2) expression. Strikingly, ALCAT1 overexpression also leads to mtDNA instability and depletion that are reminiscent of MFN2 deficiency. Accordingly, expression of MFN2 completely rescues mitochondrial fusion defect and respiratory dysfunction. Furthermore, ablation of ALCAT1 prevents mitochondrial fragmentation from oxidative stress by up-regulating MFN2 expression, mtDNA copy number, and mtDNA fidelity. Together, these findings reveal an unexpected role of CL remodeling in mitochondrial biogenesis, linking oxidative stress by ALCAT1 to mitochondrial fusion defect.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License., [Background]: Loss-of-function mutations in PTEN-induced kinase 1 (PINK1) have been linked to familial Parkinsons disease, but the underlying pathogenic mechanism remains unclear. We previously reported that loss of PINK1 impairs mitochondrial respiratory activity in mouse brains. [Results]: In this study, we investigate how loss of PINK1 impairs mitochondrial respiration using cultured primary fibroblasts and neurons. We found that intact mitochondria in PINK1−/− cells recapitulate the respiratory defect in isolated mitochondria from PINK1−/− mouse brains, suggesting that these PINK1−/− cells are a valid experimental system to study the underlying mechanisms. Enzymatic activities of the electron transport system complexes are normal in PINK1−/− cells, but mitochondrial transmembrane potential is reduced. Interestingly, the opening of the mitochondrial permeability transition pore (mPTP) is increased in PINK1−/− cells, and this genotypic difference between PINK1−/− and control cells is eliminated by agonists or inhibitors of the mPTP. Furthermore, inhibition of mPTP opening rescues the defects in transmembrane potential and respiration in PINK1−/− cells. Consistent with our earlier findings in mouse brains, mitochondrial morphology is similar between PINK1−/− and wild-type cells, indicating that the observed mitochondrial functional defects are not due to morphological changes. Following FCCP treatment, calcium increases in the cytosol are higher in PINK1−/− compared to wild-type cells, suggesting that intra-mitochondrial calcium concentration is higher in the absence of PINK1. [Conclusions]: Our findings show that loss of PINK1 causes selective increases in mPTP opening and mitochondrial calcium, and that the excessive mPTP opening may underlie the mitochondrial functional defects observed in PINK1−/− cells., This work was supported by a grant from the NINDS (R01 NS041779).

In recent years, the dynamic nature of mitochondria has been discovered to be critical for their function. Here we discuss the molecular basis of mitochondrial fusion, its protective role in neurodegeneration, and its importance in cellular function. The mitofusins Mfn1 and Mfn2, GTPases localized to the outer membrane, mediate outer-membrane fusion. OPA1, a GTPase associated with the inner membrane, mediates subsequent inner-membrane fusion. Mutations in Mfn2 or OPA1 cause neurodegenerative diseases. Mouse models with defects in mitochondrial fusion genes have provided important avenues for understanding how fusion maintains mitochondrial physiology and neuronal function. Mitochondrial fusion enables content mixing within a mitochondrial population, thereby preventing permanent loss of essential components. Cells with reduced mitochondrial fusion, as a consequence, show a subpopulation of mitochondria that lack mtDNA nucleoids. Such mtDNA defects lead to respiration-deficient mitochondria, and their accumulation in neurons leads to impaired outgrowth of cellular processes and ultimately neurodegeneration.

SummaryMitochondria are highly mobile and dynamic organelles that continually fuse and divide. These processes allow mitochondria to exchange contents, including mitochondrial DNA (mtDNA). Here we examine the functions of mitochondrial fusion in differentiated skeletal muscle through conditional deletion of the mitofusins Mfn1 and Mfn2, mitochondrial GTPases essential for fusion. Loss of the mitofusins causes severe mitochondrial dysfunction, compensatory mitochondrial proliferation, and muscle atrophy. Mutant mice have severe mtDNA depletion in muscle that precedes physiological abnormalities. Moreover, the mitochondrial genomes of the mutant muscle rapidly accumulate point mutations and deletions. In a related experiment, we find that disruption of mitochondrial fusion strongly increases mitochondrial dysfunction and lethality in a mouse model with high levels of mtDNA mutations. With its dual function in safeguarding mtDNA integrity and preserving mtDNA function in the face of mutations, mitochondrial fusion is likely to be a protective factor in human disorders associated with mtDNA mutations.PaperClip

Mitochondrial fusion requires the coordinated fusion of the outer and inner membranes. Three large GTPases—OPA1 and the mitofusins Mfn1 and Mfn2—are essential for the fusion of mammalian mitochondria. OPA1 is mutated in dominant optic atrophy, a neurodegenerative disease of the optic nerve. In yeast, the OPA1 ortholog Mgm1 is required for inner membrane fusion in vitro; nevertheless, yeast lacking Mgm1 show neither outer nor inner membrane fusion in vivo, because of the tight coupling between these two processes. We find that outer membrane fusion can be readily visualized in OPA1-null mouse cells in vivo, but these events do not progress to inner membrane fusion. Similar defects are found in cells lacking prohibitins, which are required for proper OPA1 processing. In contrast, double Mfn-null cells show neither outer nor inner membrane fusion. Mitochondria in OPA1-null cells often contain multiple matrix compartments bounded together by a single outer membrane, consistent with uncoupling of outer versus inner membrane fusion. In addition, unlike mitofusins and yeast Mgm1, OPA1 is not required on adjacent mitochondria to mediate membrane fusion. These results indicate that mammalian mitofusins and OPA1 mediate distinct sequential fusion steps that are readily uncoupled, in contrast to the situation in yeast.

International audience; Mitochondria are dynamic organelles, the morphology of which results from an equilibrium between two opposing processes, fusion and fission. Mitochondrial fusion relies on dynamin‐related GTPases, the mitofusins (MFN1 and 2) in the outer mitochondrial membrane and OPA1 (optic atrophy 1) in the inner mitochondrial membrane. Apart from a role in the maintenance of mitochondrial DNA, little is known about the physiological role of mitochondrial fusion. Here we report that mitochondria hyperfuse and form a highly interconnected network in cells exposed to selective stresses. This process precedes mitochondrial fission when it is triggered by apoptotic stimuli such as UV irradiation or actinomycin D. Stress‐induced mitochondrial hyperfusion (SIMH) is independent of MFN2, BAX/BAK, and prohibitins, but requires L‐OPA1, MFN1, and the mitochondrial inner membrane protein SLP‐2. In the absence of SLP‐2, L‐OPA1 is lost and SIMH is prevented. SIMH is accompanied by increased mitochondrial ATP production and represents a novel adaptive pro‐survival response against stress.

The ligand (KL) for the c-kit receptor is a growth factor encoded at the mouse steel (Sl) locus. KL exists in both cell surface and soluble forms, though little is known of the regulation and functional significance of these forms. We show here that tissue-specific alternative splicing gives two types of KL mRNA. Both encode a transmembrane domain, but in transfected cells one produced the soluble form of KL at relatively high levels, whereas the other preferentially gave the cell surface form. Cell surface KL not only stimulated proliferation, but also mediated cell-cell adhesion. The SId allele, which impairs development of hematopoietic cells, melanocytes, and germ cells, has a deletion in the KL gene removing the transmembrane and intracellular domains. Expression of a corresponding cDNA gave a soluble protein that stimulated cellular proliferation but was not associated with the cell surface. These results provide evidence that cell surface KL has a critical role in the intact organism.

Charcot–Marie–Tooth (CMT) disease type 2A is a progressive, neurodegenerative disorder affecting long peripheral motor and sensory nerves. The most common clinical sign is weakness in the lower legs and feet, associated with muscle atrophy and gait defects. The axonopathy in CMT2A is caused by mutations in Mitofusin 2 (Mfn2), a mitochondrial GTPase necessary for the fusion of mitochondria. Most Mfn2 disease alleles dominantly aggregate mitochondria upon expression in cultured fibroblasts and neurons. To determine whether this property is related to neuronal pathogenesis, we used the HB9 promoter to drive expression of a pathogenic allele, Mfn2^(T105M), in the motor neurons of transgenic mice. Transgenic mice develop key clinical signs of CMT2A disease in a dosage-dependent manner. They have a severe gait defect due to an inability to dorsi-flex the hindpaws. Consequently, affected animals drag their hindpaws while walking and support themselves on the hind knuckles, rather than the soles. This distal muscle weakness is associated with reduced numbers of motor axons in the motor roots and severe reduction of the anterior calf muscles. Many motor neurons from affected animals show improper mitochondrial distribution, characterized by tight clusters of mitochondria within axons. This transgenic line recapitulates key motor features of CMT2A and provides a system to dissect the function of mitochondria in the axons of mammalian motor neurons.

OPA1, a dynamin-related guanosine triphosphatase mutated in dominant optic atrophy, is required for the fusion of mitochondria. Proteolytic cleavage by the mitochondrial processing peptidase generates long isoforms from eight messenger RNA (mRNA) splice forms, whereas further cleavages at protease sites S1 and S2 generate short forms. Using OPA1-null cells, we developed a cellular system to study how individual OPA1 splice forms function in mitochondrial fusion. Only mRNA splice forms that generate a long isoform in addition to one or more short isoforms support substantial mitochondrial fusion activity. On their own, long and short OPA1 isoforms have little activity, but, when coexpressed, they functionally complement each other. Loss of mitochondrial membrane potential destabilizes the long isoforms and enhances the cleavage of OPA1 at S1 but not S2. Cleavage at S2 is regulated by the i-AAA protease Yme1L. Our results suggest that mammalian cells have multiple pathways to control mitochondrial fusion through regulation of the spectrum of OPA1 isoforms.

Mfn2, an oligomeric mitochondrial protein important for mitochondrial fusion, is mutated in Charcot-Marie-Tooth disease (CMT) type 2A, a peripheral neuropathy characterized by axonal degeneration. In addition to homooligomeric complexes, Mfn2 also associates with Mfn1, but the functional significance of such heterooligomeric complexes is unknown. Also unknown is why Mfn2 mutations in CMT2A lead to cell type–specific defects given the widespread expression of Mfn2. In this study, we show that homooligomeric complexes formed by many Mfn2 disease mutants are nonfunctional for mitochondrial fusion. However, wild-type Mfn1 complements mutant Mfn2 through the formation of heterooligomeric complexes, including complexes that form in trans between mitochondria. Wild-type Mfn2 cannot complement the disease alleles. Our results highlight the functional importance of Mfn1–Mfn2 heterooligomeric complexes and the close interplay between the two mitofusins in the control of mitochondrial fusion. Furthermore, they suggest that tissues with low Mfn1 expression are vulnerable in CMT2A and that methods to increase Mfn1 expression in the peripheral nervous system would benefit CMT2A patients.

Fusion of vesicles into target membranes during many types of regulated exocytosis requires both SNARE-complex proteins and fusogenic lipids, such as phosphatidic acid. Mitochondrial fusion is less well understood but distinct, as it is mediated instead by the protein Mitofusin (Mfn). Here, we identify an ancestral member of the phospholipase D (PLD) superfamily of lipid-modifying enzymes that is required for mitochondrial fusion. Mitochondrial PLD (MitoPLD) targets to the external face of mitochondria and promotes trans-mitochondrial membrane adherence in a Mfn-dependent manner by hydrolysing cardiolipin to generate phosphatidic acid. These findings reveal that although mitochondrial fusion and regulated exocytic fusion are mediated by distinct sets of protein machinery, the underlying processes are unexpectedly linked by the generation of a common fusogenic lipid. Moreover, our findings suggest a novel basis for the mitochondrial fragmentation observed during apoptosis.

Mitofusin 2 (Mfn2) is a mitochondrial membrane protein that participates in mitochondrial fusion and regulates mitochondrial metabolism in mammalian cells. Here, we show that Mfn2 gene expression is induced in skeletal muscle and brown adipose tissue by conditions associated with enhanced energy expenditure, such as cold exposure or beta(3)-adrenergic agonist treatment. In keeping with the role of peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1 alpha on energy expenditure, we demonstrate a stimulatory effect of PGC-1 alpha on Mfn2 mRNA and protein expression in muscle cells. PGC-1 alpha also stimulated the activity of the Mfn2 promoter, which required the integrity of estrogen-related receptor-alpha (ERR alpha)-binding elements located at -413/-398. ERR alpha also activated the transcriptional activity of the Mfn2 promoter, and the effects were synergic with those of PGC-1 alpha. Mfn2 loss of function reduced the stimulatory effect of PGC-1 alpha on mitochondrial membrane potential. Exposure to cold substantially increased Mfn2 gene expression in skeletal muscle from heterozygous Mfn2 knock-out mice, which occurred in the presence of higher levels of PGC-1 alpha mRNA compared with control mice. Our results indicate the existence of a regulatory pathway involving PGC-1 alpha, ERR alpha, and Mfn2. Alterations in this regulatory pathway may participate in the pathophysiology of insulin-resistant conditions and type 2 diabetes.

Vesicle fusion involves vesicle tethering, docking, and membrane merger. We show that mitofusin, an integral mitochondrial membrane protein, is required on adjacent mitochondria to mediate fusion, which indicates that mitofusin complexes act in trans (that is, between adjacent mitochondria). A heptad repeat region (HR2) mediates mitofusin oligomerization by assembling a dimeric, antiparallel coiled coil. The transmembrane segments are located at opposite ends of the 95 angstrom coiled coil and provide a mechanism for organelle tethering. Consistent with this proposal, truncated mitofusin, in an HR2-dependent manner, causes mitochondria to become apposed with a uniform gap. Our results suggest that HR2 functions as a mitochondrial tether before fusion.

A dynamic balance of organelle fusion and fission regulates mitochondrial morphology. During apoptosis this balance is altered, leading to an extensive fragmentation of the mitochondria. Here, we describe a novel assay of mitochondrial dynamics based on confocal imaging of cells expressing a mitochondrial matrix–targeted photoactivable green fluorescent protein that enables detection and quantification of organelle fusion in living cells. Using this assay, we visualize and quantitate mitochondrial fusion rates in healthy and apoptotic cells. During apoptosis, mitochondrial fusion is blocked independently of caspase activation. The block in mitochondrial fusion occurs within the same time range as Bax coalescence on the mitochondria and outer mitochondrial membrane permeabilization, and it may be a consequence of Bax/Bak activation during apoptosis.

Mitochondria contain their own circular genome, with mitochondria-specific transcription and replication systems and corresponding regulatory proteins. All of these proteins are encoded in the nuclear genome and are post-translationally imported into mitochondria. In addition, several nuclear transcription factors have been reported to act in mitochondria, but there has been no comprehensive mapping of their occupancy patterns and it is not clear how many other factors may also be found in mitochondria. Here we address these questions by using ChIP-seq data from the ENCODE, mouseENCODE and modENCODE consortia for 151 human, 31 mouse and 35 C. elegans factors. We identified 8 human and 3 mouse transcription factors with strong localized enrichment over the mitochondrial genome that was usually associated with the corresponding recognition sequence motif. Notably, these sites of occupancy are often the sites with highest ChIP-seq signal intensity within both the nuclear and mitochondrial genomes and are thus best explained as true binding events to mitochondrial DNA, which exist in high copy number in each cell. We corroborated these findings by immunocytochemical staining evidence for mitochondrial localization. However, we were unable to find clear evidence for mitochondrial binding in ENCODE and other publicly available ChIP-seq data for most factors previously reported to localize there. As the first global analysis of nuclear transcription factors binding in mitochondria, this work opens the door to future studies that probe the functional significance of the phenomenon.

WW domains are conserved protein motifs of 38-40 amino acids found in a broad spectrum of proteins. They mediate protein-protein interactions by binding proline-rich modules in ligands. A 10 amino acid proline-rich portion of the morphogenic protein, formin, is bound in vitro by both the WW domain of the formin-binding protein 11 (FBP11) and the SH3 domain of Abl. To explore whether the FBP11 WW domain and Abl SH3 domain bind to similar ligands, we screened a mouse limb bud expression library for putative ligands of the FBP11 WW domain. In so doing, we identified eight ligands (WBP3 through WBP10), each of which contains a proline-rich region or regions. Peptide sequence comparisons of the ligands revealed a conserved motif of 10 amino acids that acts as a modular sequence binding the FBP11 WW domain, but not the WW domain of the putative signal transducing factor, hYAP65. Interestingly, the consensus ligand for the FBP11 WW domain contains residues that are also required for binding by the Abl SH3 domain. These findings support the notion that the FBP11 WW domain and the Abl SH3 domain can compete for the same proline-rich ligands and suggest that at least two subclasses of WW domains exist, namely those that bind a PPLP motif, and those that bind a PPXY motif.

Mutations in the mouse formin (Fmn) gene, formerly known as the limb deformity (ld) gene, give rise to recessively inherited limb deformities and renal malformations or aplasia. The Fmn gene encodes many differentially processed transcripts that are expressed in both adult and embryonic tissues. To study the genomic organization of the Fmn locus, we have used Fmn probes to isolate and characterize genomic clones spanning 500 kb. Our analysis of these clones shows that the Fmn gene is composed of at least 24 exons and spans 400 kb. We have identified two novel exons that are expressed in the developing embryonic limb bud as well as adult tissues such as brain and kidney. We have also used a microsatellite polymorphism from within the Fmn gene to map it genetically to a 2.2-cM interval between D2Mit58 and D2Mit103.

Strong’s Luxoid (lstD) is a semidominant mouse mutation in which heterozygotes show preaxial hindlimb polydactyly, and homozygotes show fore- and hindlimb polydactyly. The digit patterns of these polydactylous limbs resemble those caused by polarizing grafts, since additional digits with posterior character are present at the anterior side of the limb. Such observations suggest that lstD limb buds might contain a genetically determined ectopic region of polarizing activity. Accordingly, we show that mutant embryos ectopically express the pattern-determining genes fibroblast growth factor 4 (fgf-4), sonic hedgehog (shh), and Hoxd-12 in the anterior region of the limb. Further, we show that anterior mesoderm from mutant limbs exhibits polarizing activity when grafted into host chicken limbs. In contrast to an experimentally derived polydactylous transgenic mouse, forelimbs of homozygotes show a normal pattern of Hoxb-8 expression, indicating that the duplication of polarizing tissue here occurs downstream or independently of Hoxb-8. We suggest that the lst gene product is involved in anteroposterior axis formation during normal limb development.

Mutations of the mouse limb deformity locus, ld, map to Chromosome (Chr) 2 and result in defects in the morphogenesis and patterning of the limb and kidney. Complementation studies have defined the existence of five recessive ld alleles. Remarkably, two of these, ldTgHd and ldTgBri, are transgene-induced mutations. Recovery of the first transgene insertional allele, ldTgHd, facilitated the molecular cloning of a large (greater than 200 kb) candidate gene at the ld locus. This gene is broadly transcribed and encodes a set of novel protein isoforms, termed formins. Here we present characterization of the ldTgBri mutation that supports the molecular identification of the ld gene. We show that the ldTgBri fails to complement both the ldTgHd and the ldOR alleles and that it has undergone a genomic deletion that disrupts the cloned ld gene and its transcripts. Curiously, the ldTgBri deletion encompasses the same 11-kb interval in which the ldTgHd insertion occurred and in which a chromosomal rearrangement has been identified in a third allele, ldIn2. These findings suggest that this region of the ld gene is a preferential site for illegitimate recombination.