Changes in mitochondrial morphology that occur during cell cycle, differentiation, and death are tightly regulated by the balance between fusion and fission processes. Excessive fragmentation can be caused by inhibition of the fusion machinery and is a common consequence of dysfunction of the organelle. Here, we show a role for calcineurin-dependent translocation of the profission dynamin related protein 1 (Drp1) to mitochondria in dysfunction-induced fragmentation. When mitochondrial depolarization is associated with sustained cytosolic Ca ²⁺ rise, it activates the cytosolic phosphatase calcineurin that normally interacts with Drp1. Calcineurin-dependent dephosphorylation of Drp1, and in particular of its conserved serine 637, regulates its translocation to mitochondria as substantiated by site directed mutagenesis. Thus, fragmentation of depolarized mitochondria depends on a loop involving sustained Ca ²⁺ rise, activation of calcineurin, and dephosphorylation of Drp1 and its translocation to the organelle.

Mitochondria in cells comprise a tubulovesicular reticulum shaped by dynamic fission and fusion events. The multimeric dynamin-like GTPase Drp1 is a critical protein mediating mitochondrial division. It harbors multiple motifs including GTP-binding, middle, and GTPase effector (GED) domains that are important for both intramolecular and intermolecular interactions. As for other members of the dynamin superfamily, such interactions are critical for assembly of higher-order structures and cooperative increases in GTPase activity. Although the functions of Drp1 in cells have been extensively studied, mechanisms underlying its regulation remain less clear. Here, we have identified cAMP-dependent protein kinase-dependent phosphorylation of Drp1 within the GED domain at Ser⁶³⁷ that inhibits Drp1 GTPase activity. Mechanistically, this change in GTPase activity likely derives from decreased interaction of GTP-binding/middle domains with the GED domain since the phosphomimetic S637D mutation impairs this intramolecular interaction but not Drp1-Drp1 intermolecular interactions. Using the phosphomimetic S637D substitution, we also demonstrate that mitochondrial fission is prominently inhibited in cells. Thus, protein phosphorylation at Ser⁶³⁷ results in clear alterations in Drp1 function and mitochondrial morphology that are likely involved in dynamic regulation of mitochondrial division in cells.

Mitochondrial dysfunction in skeletal muscle has been implicated in the development of insulin resistance and type 2 diabetes. Considering the importance of mitochondrial dynamics in mitochondrial and cellular functions, we hypothesized that obesity and excess energy intake shift the balance of mitochondrial dynamics, further contributing to mitochondrial dysfunction and metabolic deterioration in skeletal muscle. First, we revealed that excess palmitate (PA), but not hyperglycemia, hyperinsulinemia, or elevated tumor necrosis factor alpha, induced mitochondrial fragmentation and increased mitochondrion-associated Drp1 and Fis1 in differentiated C2C12 muscle cells. This fragmentation was associated with increased oxidative stress, mitochondrial depolarization, loss of ATP production, and reduced insulin-stimulated glucose uptake. Both genetic and pharmacological inhibition of Drp1 attenuated PA-induced mitochondrial fragmentation, mitochondrial depolarization, and insulin resistance in C2C12 cells. Furthermore, we found smaller and shorter mitochondria and increased mitochondrial fission machinery in the skeletal muscle of mice with genetic obesity and those with diet-induced obesity. Inhibition of mitochondrial fission improved the muscle insulin signaling and systemic insulin sensitivity of obese mice. Our findings indicated that aberrant mitochondrial fission is causally associated with mitochondrial dysfunction and insulin resistance in skeletal muscle. Thus, disruption of mitochondrial dynamics may underlie the pathogenesis of muscle insulin resistance in obesity and type 2 diabetes.

Dynamin‐related protein (Drp) 1 is a key regulator of mitochondrial fission and is composed of GTP‐binding, Middle, insert B, and C‐terminal GTPase effector (GED) domains. Drpl associates with mitochondrial fission sites and promotes membrane constriction through its intrinsic GTPase activity. The mechanisms that regulate Drpl activity remain poorly understood but are likely to involve reversible post‐translational modifications, such as conjugation of small ubiquitin‐like modifier (SUMO) proteins. Through a detailed analysis, we find that Drpl interacts with the SUMO‐conjugating enzyme Ubc9 via multiple regions and demonstrate that Drpl is a direct target of SUMO modification by all three SUMO isoforms. While Drpl does not harbor consensus SUMOylation sequences, our analysis identified2 clusters of lysine residues within the B domain that serve as noncanonical conjugation sites. Although initial analysis indicates that mitochondrial recruitment of ectopically expressed Drpl in response to staurosporine is unaffected by loss of SUMOylation, we find that Drpl SUMOylation is enhanced in the context of the K38A mutation. This dominant‐negative mutant, which is deficient in GTP binding and hydrolysis, does not associate with mitochondria and prevents normal mitochondrial fission. This finding suggests that SUMOylation of Drpl is linked to its activity cycle and is influenced by Drpl localization.—Figueroa‐Romero, C., Iniguez‐Lluhi, J. A., Stadler, J., Chang, C.‐R., Arnoult, D., Keller, P. J., Hong, Y., Blackstone, C., Feldman, E. L. SUMOylation of the mitochondrial fission protein Drpl occurs at multiple nonconsensus sites within the B domain and is linked to its activity cycle. FASEB J. 23, 3917–3927 (2009). www.fasebj.org

Mitochondria dynamically fuse and divide within cells, and the proper balance of fusion and fission is necessary for normal mitochondrial function, morphology, and distribution. Drp1 is a dynamin-related GTPase required for mitochondrial fission in mammalian cells. It harbors four distinct domains: GTP-binding, middle, insert B, and GTPase effector. A lethal mutation (A395D) within the Drp1 middle domain was reported in a neonate with microcephaly, abnormal brain development, optic atrophy, and lactic acidemia (Waterham, H. R., Koster, J., van Roermund, C. W., Mooyer, P. A., Wanders, R. J., and Leonard, J. V. (2007) N. Engl. J. Med. 356, 1736-1741). Mitochondria within patient-derived fibroblasts were markedly elongated, but the molecular mechanisms underlying these findings were not demonstrated. Because the middle domain is particularly important for the self-assembly of some dynamin superfamily proteins, we tested the hypothesis that this A395D mutation, and two other middle domain mutations (G350D, G363D) were important for Drp1 tetramerization, higher order assembly, and function. Although tetramerization appeared largely intact, each of these mutations compromised higher order assembly and assembly-dependent stimulation of Drp1 GTPase activity. Moreover, mutant Drp1 proteins exhibited impaired localization to mitochondria, indicating that this higher order assembly is important for mitochondrial recruitment, retention, or both. Overexpression of these middle domain mutants markedly inhibited mitochondrial division in cells. Thus, the Drp1 A395D lethal defect likely resulted in impaired higher order assembly of Drp1 at mitochondria, leading to decreased fission, elongated mitochondria, and altered cellular distribution of mitochondria.