While the dynamin GTPase Drp1 plays a critical role during mitochondrial fission, mechanisms controlling its recruitment to fission sites are unclear. A current assumption is that cytosolic Drp1 is recruited directly to fission sites immediately prior to fission. Using live-cell microscopy, we find evidence for a different model, progressive maturation of Drp1 oligomers on mitochondria through incorporation of smaller mitochondrially-bound Drp1 units. Maturation of a stable Drp1 oligomer does not forcibly lead to fission. Drp1 oligomers also translocate directionally along mitochondria. Ionomycin, a calcium ionophore, causes rapid mitochondrial accumulation of actin filaments followed by Drp1 accumulation at the fission site, and increases fission rate. Inhibiting actin polymerization, myosin IIA, or the formin INF2 reduces both un-stimulated and ionomycin-induced Drp1 accumulation and mitochondrial fission. Actin filaments bind purified Drp1 and increase GTPase activity in a manner that is synergistic with the mitochondrial protein Mff, suggesting a role for direct Drp1/actin interaction. We propose that Drp1 is in dynamic equilibrium on mitochondria in a fission-independent manner, and that fission factors such as actin filaments target productive oligomerization to fission sites.DOI: http://dx.doi.org/10.7554/eLife.11553.001

The mitochondrial signaling complex PKA/AKAP1 protects neurons against mitochondrial fragmentation and cell death by phosphorylating and inactivating the mitochondrial fission enzyme Drp1.

The neurodegenerative disorder spinocerebellar ataxia 12 (SCA12) is caused by CAG repeat expansion in the non-coding region of the PPP2R2B gene. PPP2R2B encodes B␤1 and B␤2, alternatively spliced and neuron-specific regulatory subunits of the protein phosphatase 2A (PP2A) holoenzyme. We show here that in PC12 cells and hippocampal neurons, cell stressors induced a rapid translocation of PP2A/B␤2 to mitochondria to promote apoptosis. Conversely, silencing of PP2A/B␤2 protected hippocampal neurons against free radical-mediated, excitotoxic, and ischemic insults. Evidence is accumulating that the mitochondrial fission/fusion equilibrium is an important determinant of cell survival. Accordingly, we found that B␤2 expression induces mitochondrial fragmentation, whereas B␤2 silencing or inhibition resulted in mitochondrial elongation. Based on epistasis experiments involving Bcl2 and core components of the mitochondrial fission machinery (Fis1 and dynamin-related protein 1), mitochondrial fragmentation occurs upstream of apoptosis and is both necessary and sufficient for hippocampal neuron death. Our data provide the first example of a proapoptotic phosphatase that predisposes to neuronal death by promoting mitochondrial division and point to a possible imbalance of the mitochondrial morphogenetic equilibrium in the pathogenesis of SCA12.Mitochondrial morphology and assembly of mitochondria into a contiguous network is a consequence of opposing fission and fusion events (1-3). In neurons, physiological levels of mitochondrial fission are necessary for axonal and dendritic transport of mitochondria and the proper development and function of synapses (4, 5). However, mitochondrial fragmentation is also an integral aspect of apoptosis because enhancing and inhibiting fission can promote and delay apoptosis, respectively (6, 7). Mitochondrial fission and fusion are separate processes catalyzed by large GTPases that were initially identified in yeast (8). The principal fission enzyme is dynamin-related protein 1 (Drp1), 5 which, in analogy to the endocytosis motor dynamin, is thought to utilize GTP hydrolysis to mechanically constrict and sever mitochondria. Drp1 is recruited from the cytosol to the outer mitochondrial membrane (OMM) by a multiprotein complex that includes Fis1. In mammals, outer mitochondrial membrane fusion is carried out by two transmembrane GTPases, mitofusin 1 and 2 (Mfn1/2), which act in concert with the inner membrane fusion enzyme optic atrophy 1 (Opa1) (8).The fundamental importance of a healthy mitochondrial fission/fusion balance in neurons is documented by the discovery that two common neurodegenerative disorders, CharcotMarie-Tooth disease type 2A and dominant optic atrophy, are caused by mutations in mitochondrial fusion GTPases, Mfn2 and Opa1, respectively (9 -11). Similarly, a dominant-inactivating mutation in Drp1 was recently linked to microcephaly and other neurological birth defects (12). Despite the clear relevance of mitochondrial morphogenesis to neuronal survival (6, 13-15), we know...

Actin stimulates oligomerization and mitochondrial accumulation of Drp1, a mitochondrial fission protein. Drp1 binds actin filaments in an unusually dynamic manner that is strongly influenced by guanine nucleotide.

Mitochondrial fission and fusion impact numerous cellular functions and neurons are particularly sensitive to perturbations in mitochondrial dynamics. Here we describe that male mice lacking the mitochondrial A-kinase anchoring protein 1 (AKAP1) exhibit increased sensitivity in the transient middle cerebral artery occlusion model of focal ischemia. At the ultrastructural level, AKAP1 mice have smaller mitochondria and increased contacts between mitochondria and the endoplasmic reticulum in the brain. Mechanistically, deletion of AKAP1 dysregulates complex II of the electron transport chain, increases superoxide production, and impairs Ca homeostasis in neurons subjected to excitotoxic glutamate. Ca deregulation in neurons lacking AKAP1 can be attributed to loss of inhibitory phosphorylation of the mitochondrial fission enzyme dynamin-related protein 1 (Drp1) at the protein kinase A (PKA) site Ser637. Our results indicate that inhibition of Drp1-dependent mitochondrial fission by the outer mitochondrial AKAP1/PKA complex protects neurons from ischemic stroke by maintaining respiratory chain activity, inhibiting superoxide production, and delaying Ca deregulation. They also provide the first genetic evidence that Drp1 inhibition may be of therapeutic relevance for the treatment of stroke and neurodegeneration. Previous work suggests that activation of dynamin-related protein 1 (Drp1) and mitochondrial fission contribute to ischemic injury in the brain. However, the specificity and efficacy of the pharmacological Drp1 inhibitor mdivi-1 that was used has now been discredited by several high-profile studies. Our report is timely and highly impactful because it provides the first evidence that genetic disinhibition of Drp1 via knock-out of the mitochondrial protein kinase A (PKA) scaffold AKAP1 exacerbates stroke injury in mice. Mechanistically, we show that electron transport deficiency, increased superoxide production, and Ca overload result from genetic disinhibition of Drp1. In summary, our work settles current controversies regarding the role of mitochondrial fission in neuronal injury, provides mechanisms, and suggests that fission inhibitors hold promise as future therapeutic agents.