[Abstract unavailable

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. Calcineurindependent 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.fission ͉ phosphatase ͉ subcellular localization ͉ calcium ͉ cyclosporine A

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 GTPbinding, 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 Ser 637 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 Ser 637 results in clear alterations in Drp1 function and mitochondrial morphology that are likely involved in dynamic regulation of mitochondrial division in cells.Mitochondria are critical for a number of cellular functions because of their roles in producing energy, buffering calcium, and regulating apoptosis. In contrast to their common depiction as sausage-shaped organelles in schematic diagrams, they form a dynamic reticulum in the cell, continuously dividing and fusing with one another (1-4). The steady-state equilibrium of these opposing processes is altered during critical cellular events such as apoptosis and cell division, and perturbation of this balance has been directly implicated in a number of inherited neurological disorders including Charcot-Marie-Tooth neuropathy and optic atrophy type 1 (3, 5) as well as a neonatal lethal syndrome of microcephaly, abnormal brain development, optic atrophy, and lactic acidemia (6).The dynamin-related GTPase Drp1 is an evolutionarily conserved protein that mediates mitochondrial division, and its functional impairment results in aggregates of large, interconnected mitochondria within cells. Drp1 is also involved in the mitochondrial scissioning that occurs during apoptosis and cell division (5, 7). However, mechanisms by which Drp1 function is regulated during such critical cellular events are less well understood. Frequently, cellular responses to changing conditions are mediated by reversible covalent modifications of existing molecules, and Drp1 is modified post-translationally by protein phosphorylation, ubiquitylation, and sumoylation (7-10). However, in most cases, neither the direct impact of these modifications on Drp1 functions nor the sites of modification have been thoroughly investigated.Like many dynamin-related proteins, Drp1 ...

Hereditary spastic paraplegias (HSPs; SPG1-45) are inherited neurological disorders characterized by lower extremity spastic weakness. More than half of HSP cases result from autosomal dominant mutations in atlastin-1 (also known as SPG3A), receptor expression enhancing protein 1 (REEP1; SPG31), or spastin (SPG4). The atlastin-1 GTPase interacts with spastin, a microtubule-severing ATPase, as well as with the DP1/Yop1p and reticulon families of ER-shaping proteins, and SPG3A caused by atlastin-1 mutations has been linked pathogenically to abnormal ER morphology. Here we investigated SPG31 by analyzing the distribution, interactions, and functions of REEP1. We determined that REEP1 is structurally related to the DP1/Yop1p family of ER-shaping proteins and localizes to the ER in cultured rat cerebral cortical neurons, where it colocalizes with spastin and atlastin-1. Upon overexpression in COS7 cells, REEP1 formed protein complexes with atlastin-1 and spastin within the tubular ER, and these interactions required hydrophobic hairpin domains in each of these proteins. REEP proteins were required for ER network formation in vitro, and REEP1 also bound microtubules and promoted ER alignment along the microtubule cytoskeleton in COS7 cells. A SPG31 mutant REEP1 lacking the C-terminal cytoplasmic region did not interact with microtubules and disrupted the ER network. These data indicate that the HSP proteins atlastin-1, spastin, and REEP1 interact within the tubular ER membrane in corticospinal neurons to coordinate ER shaping and microtubule dynamics. Thus, defects in tubular ER shaping and network interactions with the microtubule cytoskeleton seem to be the predominant pathogenic mechanism of HSP.