GBF1 and Arf1 interact with Miro and regulate mitochondrial positioning within cells.
ABSTRACT: The spatial organization of cells depends on coordination between cytoskeletal systems and intracellular organelles. The Arf1 small G protein and its activator GBF1 are important regulators of Golgi organization, maintaining its morphology and function. Here we show that GBF1 and its substrate Arf1 regulate the spatial organization of mitochondria in a microtubule-dependent manner. Miro is a mitochondrial membrane protein that interacts through adaptors with microtubule motor proteins such as cytoplasmic dynein, the major microtubule minus end directed motor. We demonstrate a physical interaction between GBF1 and Miro, and also between the active GTP-bound form of Arf1 and Miro. Inhibition of GBF1, inhibition of Arf1 activation, or overexpression of Miro, caused a collapse of the mitochondrial network towards the centrosome. The change in mitochondrial morphology upon GBF1 inhibition was due to a two-fold increase in the time engaged in retrograde movement compared to control conditions. Electron tomography revealed that GBF1 inhibition also resulted in larger mitochondria with more complex morphology. Miro silencing or drug inhibition of cytoplasmic dynein activity blocked the GBF1-dependent repositioning of mitochondria. Our results show that blocking GBF1 function promotes dynein- and Miro-dependent retrograde mitochondrial transport along microtubules towards the microtubule-organizing center, where they form an interconnected network.
Project description:Microtubule-based transport of mitochondria into dendrites and axons is vital for sustaining neuronal function. Transport along microtubule tracks proceeds in a series of plus and minus end-directed movements that are facilitated by kinesin and dynein motors. How the opposing movements are controlled to achieve effective transport over large distances remains unclear. Previous studies showed that the conserved mitochondrial GTPase Miro is required for mitochondrial transport into axons and dendrites and serves as a Ca(2+) sensor that controls mitochondrial mobility. To directly examine Miro's significance for kinesin- and/or dynein-mediated mitochondrial motility, we live-imaged movements of GFP-tagged mitochondria in larval Drosophila motor axons upon genetic manipulations of Miro. Loss of Drosophila Miro (dMiro) reduced the effectiveness of both anterograde and retrograde mitochondrial transport by selectively impairing kinesin- or dynein-mediated movements, depending on the direction of net transport. Net anterogradely transported mitochondria exhibited reduced kinesin- but normal dynein-mediated movements. Net retrogradely transported mitochondria exhibited much shorter dynein-mediated movements, whereas kinesin-mediated movements were minimally affected. In both cases, the duration of short stationary phases increased proportionally. Overexpression (OE) of dMiro also impaired the effectiveness of mitochondrial transport. Finally, loss and OE of dMiro altered the length of mitochondria in axons through a mechanistically separate pathway. We suggest that dMiro promotes effective antero- and retrograde mitochondrial transport by extending the processivity of kinesin and dynein motors according to a mitochondrion's programmed direction of transport.
Project description:The small GTPase Arf1 plays critical roles in membrane traffic by initiating the recruitment of coat proteins and by modulating the activity of lipid-modifying enzymes. Here, we report an unexpected but evolutionarily conserved role for Arf1 and the ArfGEF GBF1 at mitochondria. Loss of function of ARF-1 or GBF-1 impaired mitochondrial morphology and activity in Caenorhabditis elegans. Similarly, mitochondrial defects were observed in mammalian and yeast cells. In Saccharomyces cerevisiae, aberrant clusters of the mitofusin Fzo1 accumulated in arf1-11 mutants and were resolved by overexpression of Cdc48, an AAA-ATPase involved in ER and mitochondria-associated degradation processes. Yeast Arf1 co-fractionated with ER and mitochondrial membranes and interacted genetically with the contact site component Gem1. Furthermore, similar mitochondrial abnormalities resulted from knockdown of either GBF-1 or contact site components in worms, suggesting that the role of Arf1 in mitochondrial functioning is linked to ER-mitochondrial contacts. Thus, Arf1 is involved in mitochondrial homeostasis and dynamics, independent of its role in vesicular traffic.
Project description:The recruitment of leukocytes to sites of inflammation is crucial for a functional immune response. In the present work, we explored the role of mitochondria in lymphocyte adhesion, polarity, and migration. We show that during adhesion to the activated endothelium under physiological flow conditions, lymphocyte mitochondria redistribute to the adhesion zone together with the microtubule-organizing center (MTOC) in an integrin-dependent manner. Mitochondrial redistribution and efficient lymphocyte adhesion to the endothelium require the function of Miro-1, an adaptor molecule that couples mitochondria to microtubules. Our data demonstrate that Miro-1 associates with the dynein complex. Moreover, mitochondria accumulate around the MTOC in response to the chemokine CXCL12/SDF-1?; this redistribution is regulated by Miro-1. CXCL12-dependent cell polarization and migration are reduced in Miro-1-silenced cells, due to impaired myosin II activation at the cell uropod and diminished actin polymerization. These data point to a key role of Miro-1 in the control of lymphocyte adhesion and migration through the regulation of mitochondrial redistribution.
Project description:Although chromosome partitioning during mitosis is well studied, the molecular mechanisms that allow proper segregation of cytoplasmic organelles in human cells are poorly understood. Here we show that mitochondria interact with growing microtubule tips and are transported towards the daughter cell periphery at the end of mitosis. This phenomenon is promoted by the direct and cell cycle-dependent interaction of the mitochondrial protein Miro and the cytoskeletal-associated protein Cenp-F. Cenp-F is recruited to mitochondria by Miro at the time of cytokinesis and associates with microtubule growing tips. Cells devoid of Cenp-F or Miro show decreased spreading of the mitochondrial network as well as cytokinesis-specific defects in mitochondrial transport towards the cell periphery. Thus, Miro and Cenp-F promote anterograde mitochondrial movement and proper mitochondrial distribution in daughter cells.
Project description:In the current model of mitochondrial trafficking, Miro1 and Miro2 Rho-GTPases regulate mitochondrial transport along microtubules by linking mitochondria to kinesin and dynein motors. By generating Miro1/2 double-knockout mouse embryos and single- and double-knockout embryonic fibroblasts, we demonstrate the essential and non-redundant roles of Miro proteins for embryonic development and subcellular mitochondrial distribution. Unexpectedly, the TRAK1 and TRAK2 motor protein adaptors can still localise to the outer mitochondrial membrane to drive anterograde mitochondrial motility in Miro1/2 double-knockout cells. In contrast, we show that TRAK2-mediated retrograde mitochondrial transport is Miro1-dependent. Interestingly, we find that Miro is critical for recruiting and stabilising the mitochondrial myosin Myo19 on the mitochondria for coupling mitochondria to the actin cytoskeleton. Moreover, Miro depletion during PINK1/Parkin-dependent mitophagy can also drive a loss of mitochondrial Myo19 upon mitochondrial damage. Finally, aberrant positioning of mitochondria in Miro1/2 double-knockout cells leads to disruption of correct mitochondrial segregation during mitosis. Thus, Miro proteins can fine-tune actin- and tubulin-dependent mitochondrial motility and positioning, to regulate key cellular functions such as cell proliferation.
Project description:Mitochondrial movements are tightly controlled to maintain energy homeostasis and prevent oxidative stress. Miro is an outer mitochondrial membrane protein that anchors mitochondria to microtubule motors and is removed to stop mitochondrial motility as an early step in the clearance of dysfunctional mitochondria. Here, using human induced pluripotent stem cell (iPSC)-derived neurons and other complementary models, we build on a previous connection of Parkinson's disease (PD)-linked PINK1 and Parkin to Miro by showing that a third PD-related protein, LRRK2, promotes Miro removal by forming a complex with Miro. Pathogenic LRRK2G2019S disrupts this function, delaying the arrest of damaged mitochondria and consequently slowing the initiation of mitophagy. Remarkably, partial reduction of Miro levels in LRRK2G2019S human neuron and Drosophila PD models rescues neurodegeneration. Miro degradation and mitochondrial motility are also impaired in sporadic PD patients. We reveal that prolonged retention of Miro, and the downstream consequences that ensue, may constitute a central component of PD pathogenesis.
Project description:Mitochondria are mobile organelles and cells regulate mitochondrial movement in order to meet the changing energy needs of each cellular region. Ca(2+) signaling, which halts both anterograde and retrograde mitochondrial motion, serves as one regulatory input. Anterograde mitochondrial movement is generated by kinesin-1, which interacts with the mitochondrial protein Miro through an adaptor protein, milton. We show that kinesin is present on all axonal mitochondria, including those that are stationary or moving retrograde. We also show that the EF-hand motifs of Miro mediate Ca(2+)-dependent arrest of mitochondria and elucidate the regulatory mechanism. Rather than dissociating kinesin-1 from mitochondria, Ca(2+)-binding permits Miro to interact directly with the motor domain of kinesin-1, preventing motor/microtubule interactions. Thus, kinesin-1 switches from an active state in which it is bound to Miro only via milton, to an inactive state in which direct binding to Miro prevents its interaction with microtubules. Disrupting Ca(2+)-dependent regulation diminishes neuronal resistance to excitotoxicity.
Project description:Mitochondria are dynamic organelles that are essential for cellular metabolism but can be functionally disrupted during pathogen infection. In neurons, mitochondria are transported on microtubules via the molecular motors kinesin-1 and dynein and recruited to energy-requiring regions such as synapses. Previous studies showed that proteins from pseudorabies virus (PRV), an alphaherpesvirus, localize to mitochondria and affect mitochondrial function. We show that PRV and herpes simplex virus type 1 (HSV-1) infection of rodent superior cervical ganglion (SCG) neurons disrupts mitochondrial motility and morphology. During PRV infection, glycoprotein B (gB)-dependent fusion events result in electrical coupling of neurons and increased action potential firing rates. Consequently, intracellular [Ca(2+)] increases and alters mitochondrial dynamics through a mechanism involving the Ca(2+)-sensitive cellular protein Miro and reduced recruitment of kinesin-1 to mitochondria. This disruption in mitochondrial dynamics is required for efficient growth and spread of PRV, indicating that altered mitochondrial transport enhances alphaherpesvirus pathogenesis and infection.
Project description:Calcium oscillations suppress mitochondrial movements along the microtubules to support on-demand distribution of mitochondria. To activate this mechanism, Ca(2+) targets a yet unidentified cytoplasmic factor that does not seem to be a microtubular motor or a kinase/phosphatase. Here, we have studied the dependence of mitochondrial dynamics on the Miro GTPases that reside in the mitochondria and contain two EF-hand Ca(2+)-binding domains, in H9c2 cells and primary neurons. At resting cytoplasmic [Ca(2+)] ([Ca(2+)](c)), movements of the mitochondria were enhanced by Miro overexpression irrespective of the presence of the EF-hands. The Ca(2+)-induced arrest of mitochondrial motility was also promoted by Miro overexpression and was suppressed when either the Miro were depleted or their EF-hand was mutated. Miro also enhanced the fusion state of the mitochondria at resting [Ca(2+)](c) but promoted mitochondrial fragmentation at high [Ca(2+)](c). These effects of Miro on mitochondrial morphology seem to involve Drp1 suppression and activation, respectively. In primary neurons, Miro also caused an increase in dendritic mitochondrial mass and enhanced mitochondrial calcium signaling. Thus, Miro proteins serve as a [Ca(2+)](c)-sensitive switch and bifunctional regulator for both the motility and fusion-fission dynamics of the mitochondria.
Project description:OBJECTIVE:The main objective of this study is to define the mechanisms by which mitochondria control vascular smooth muscle cell (VSMC) migration and impact neointimal hyperplasia. APPROACH AND RESULTS:The multifunctional CaMKII (Ca2+/calmodulin-dependent kinase II) in the mitochondrial matrix of VSMC drove a feed-forward circuit with the mitochondrial Ca2+ uniporter (MCU) to promote matrix Ca2+ influx. MCU was necessary for the activation of mitochondrial CaMKII (mtCaMKII), whereas mtCaMKII phosphorylated MCU at the regulatory site S92 that promotes Ca2+ entry. mtCaMKII was necessary and sufficient for platelet-derived growth factor-induced mitochondrial Ca2+ uptake. This effect was dependent on MCU. mtCaMKII and MCU inhibition abrogated VSMC migration and mitochondrial translocation to the leading edge. Overexpression of wild-type MCU, but not MCU S92A, mutant in MCU-/- VSMC rescued migration and mitochondrial mobility. Inhibition of microtubule, but not of actin assembly, blocked mitochondrial mobility. The outer mitochondrial membrane GTPase Miro-1 promotes mitochondrial mobility via microtubule transport but arrests it in subcellular domains of high Ca2+ concentrations. In Miro-1-/- VSMC, mitochondrial mobility and VSMC migration were abolished, and overexpression of mtCaMKII or a CaMKII inhibitory peptide in mitochondria (mtCaMKIIN) had no effect. Consistently, inhibition of mtCaMKII increased and prolonged cytosolic Ca2+ transients. mtCaMKII inhibition diminished phosphorylation of focal adhesion kinase and myosin light chain, leading to reduced focal adhesion turnover and cytoskeletal remodeling. In a transgenic model of selective mitochondrial CaMKII inhibition in VSMC, neointimal hyperplasia was significantly reduced after vascular injury. CONCLUSIONS:These findings identify mitochondrial CaMKII as a key regulator of mitochondrial Ca2+ uptake via MCU, thereby controlling mitochondrial translocation and VSMC migration after vascular injury.