Mitochondrial targeting of adenomatous polyposis coli protein is stimulated by truncating cancer mutations: regulation of Bcl-2 and implications for cell survival

The adenomatous polyposis coli (APC) protein tumor suppressor is mutated in the majority of colon cancers. Most APC gene mutations cause deletion of the C terminus and disrupt APC regulation of beta-catenin turnover, microtubule dynamics, and chromosome segregation. Truncated APC mutant peptides may also gain unique properties, not exhibited by wild-type APC, which contribute to tumor cell survival and proliferation. Here we report a differential subcellular localization pattern for wild-type and mutant APC. A pool of APC truncation mutants was detected at mitochondria by cellular fractionation and confocal microscopy. In contrast, wild-type APC located poorly at mitochondria. Similar results were observed for endogenous and stably induced forms of APC, with the shortest N-terminal mutant peptides (N750, N853, N1309, N1337) displaying the strongest mitochondrial staining. The knock down of mutant APC(N1337) in SW480 tumor cells caused an increase in apoptosis and mitochondrial membrane permeability, and this correlated with reduced Bcl-2 protein levels in mitochondrial fractions. Interestingly, the silencing of APC did not alter expression of beta-catenin or the apoptotic regulatory factors Bax, Bcl-xL, or survivin. APC formed a complex with Bcl-2 in mitochondrial fractions, and this may contribute to the APC-dependent regulation of Bcl-2. We propose that a subset of cancer mutations induce APC mitochondrial localization and that APC regulation of Bcl-2 at mitochondria may contribute to tumor cell survival.     

The Ras related GTPase Miro is not required for mitochondrial transport in Dictyostelium discoideum

Ras-related GTPases of the Miro family have been implicated in mitochondrial homeostasis and microtubule-dependent transport. They consist of two GTP-binding domains separated by calcium-binding motifs and of a C-terminal transmembrane domain that targets the protein to the outer mitochondrial membrane. We disrupted the single Miro-encoding gene in Dictyostelium discoideum and observed a substantial growth defect that we attribute to a decreased mitochondrial mass and cellular ATP content. However, mutant cells even showed an increased rate of oxygen consumption, while glucose consumption, mitochondrial transmembrane potential and production of reactive oxygen species were unaltered. Processes characteristic of the multicellular stage of the D. discoideum life cycle were also unaltered. Although mitochondria occasionally use microtubules for transport in D. discoideum, their size and distribution were not visibly affected. We found Miro in all branches of the eukaryotic tree with the exception of a few protist lineages (mainly those lacking typical mitochondria). Trypanosomatids and ciliates possess structurally unique homologs lacking the N-terminal or the C-terminal GTPase domain, respectively. We propose that in D. discoideum, as in yeasts and plants, Miro plays roles in mitochondrial homeostasis, but the ability to build a complex that regulates its association to kinesin for microtubule-dependent transport probably arose in metazoans.     

Lysine 27 Ubiquitination of the Mitochondrial Transport Protein Miro Is Dependent on Serine 65 of the Parkin Ubiquitin Ligase

Mitochondrial transport plays an important role in matching mitochondrial distribution to localized energy production and calcium buffering requirements. Here, we demonstrate that Miro1, an outer mitochondrial membrane (OMM) protein crucial for the regulation of mitochondrial trafficking and distribution, is a substrate of the PINK1/Parkin mitochondrial quality control system in human dopaminergic neuroblastoma cells. Moreover, Miro1 turnover on damaged mitochondria is altered in Parkinson disease (PD) patient-derived fibroblasts containing a pathogenic mutation in the PARK2 gene (encoding Parkin). By analyzing the kinetics of Miro1 ubiquitination, we further demonstrate that mitochondrial damage triggers rapid (within minutes) and persistent Lys-27-type ubiquitination of Miro1 on the OMM, dependent on PINK1 and Parkin. Proteasomal degradation of Miro1 is then seen on a slower time scale, within 2–3 h of the onset of ubiquitination. We find Miro ubiquitination in dopaminergic neuroblastoma cells is independent of Miro1 phosphorylation at Ser-156 but is dependent on the recently identified Ser-65 residue within Parkin that is phosphorylated by PINK1. Interestingly, we find that Miro1 can stabilize phospho-mutant versions of Parkin on the OMM, suggesting that Miro is also part of a Parkin receptor complex. Moreover, we demonstrate that Ser-65 in Parkin is critical for regulating Miro levels upon mitochondrial damage in rodent cortical neurons. Our results provide new insights into the ubiquitination-dependent regulation of the Miro-mediated mitochondrial transport machinery by PINK1/Parkin and also suggest that disruption of this regulation may be implicated in Parkinson disease pathogenesis.     

Miro proteins coordinate microtubule‐ and actin‐dependent mitochondrial transport and distribution

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.     

VPS13D bridges the ER to mitochondria and peroxisomes via Miro

Mitochondria, which are excluded from the secretory pathway, depend on lipid transport proteins for their lipid supply from the ER, where most lipids are synthesized. In yeast, the outer mitochondrial membrane GTPase Gem1 is an accessory factor of ERMES, an ER–mitochondria tethering complex that contains lipid transport domains and that functions, partially redundantly with Vps13, in lipid transfer between the two organelles. In metazoa, where VPS13, but not ERMES, is present, the Gem1 orthologue Miro was linked to mitochondrial dynamics but not to lipid transport. Here we show that Miro, including its peroxisome-enriched splice variant, recruits the lipid transport protein VPS13D, which in turn binds the ER in a VAP-dependent way and thus could provide a lipid conduit between the ER and mitochondria. These findings reveal a so far missing link between function(s) of Gem1/Miro in yeast and higher eukaryotes, where Miro is a Parkin substrate, with potential implications for Parkinson’s disease pathogenesis.     

Moving mitochondria: establishing distribution of an essential organelle

Mitochondria form a dynamic network responsible for energy production, calcium homeostasis and cell signaling. Appropriate distribution of the mitochondrial network contributes to organelle function and is essential for cell survival. Highly polarized cells, including neurons and budding yeast, are particularly sensitive to defects in mitochondrial movement and have emerged as model systems for studying mechanisms that regulate organelle distribution. Mitochondria in multicellular eukaryotes move along microtubule tracks. Actin, the primary cytoskeletal component used for transport in yeast, has more subtle functions in other organisms. Kinesin, dynein and myosin isoforms drive motor-based movement along cytoskeletal tracks. Milton and syntabulin have recently been identified as potential organelle-specific adaptor molecules for microtubule-based motors. Miro, a conserved GTPase, may function with Milton to regulate transport. In yeast, Mmr1p and Ypt11p, a Rab GTPase, are implicated in myosin V-based mitochondrial movement. These potential adaptors could regulate motor activity and therefore determine individual organelle movements. Anchoring of stationary mitochondria also contributes to organelle retention at specific sites in the cell. Together, movement and anchoring ultimately determine mitochondrial distribution throughout the cell.     

C. elegans dynamin-related protein DRP-1 controls severing of the mitochondrial outer membrane

Little is known about the mechanism of mitochondrial division. We show here that mitochondria are disrupted by mutations in a C. elegans dynamin-related protein (DRP-1). Mutant DRP-1 causes the mitochondrial matrix to retract into large blebs that are both surrounded and connected by tubules of outer membrane. This indicates that scission of the mitochondrial outer membrane is inhibited, while scission of the inner membrane still occurs. Overexpressed wild-type DRP-1 causes mitochondria to become excessively fragmented, consistent with an active role in mitochondrial scission. DRP-1 fused to GFP is observed in spots on mitochondria where scission eventually occurs. These data indicate that wild-type DRP-1 contributes to the final stages of mitochondrial division by controlling scission of the mitochondrial outer membrane.     

The GTPase dMiro is required for axonal transport of mitochondria to Drosophila synapses

We have identified EMS-induced mutations in Drosophila Miro (dMiro), an atypical mitochondrial GTPase that is orthologous to human Miro (hMiro). Mutant dmiro animals exhibit defects in locomotion and die prematurely. Mitochondria in dmiro mutant muscles and neurons are abnormally distributed. Instead of being transported into axons and dendrites, mitochondria accumulate in parallel rows in neuronal somata. Mutant neuromuscular junctions (NMJs) lack presynaptic mitochondria, but neurotransmitter release and acute Ca2+ buffering is only impaired during prolonged stimulation. Neuronal, but not muscular, expression of dMiro in dmiro mutants restored viability, transport of mitochondria to NMJs, the structure of synaptic boutons, the organization of presynaptic microtubules, and the size of postsynaptic muscles. In addition, gain of dMiro function causes an abnormal accumulation of mitochondria in distal synaptic boutons of NMJs. Together, our findings suggest that dMiro is required for controlling anterograde transport of mitochondria and their proper distribution within nerve terminals.     

Trafficking kinesin protein (TRAK)-mediated transport of mitochondria in axons of hippocampal neurons

In neurons, the proper distribution of mitochondria is essential because of a requirement for high energy and calcium buffering during synaptic neurotransmission. The efficient, regulated transport of mitochondria along axons to synapses is therefore crucial for maintaining function. The trafficking kinesin protein (TRAK)/Milton family of proteins comprises kinesin adaptors that have been implicated in the neuronal trafficking of mitochondria via their association with the mitochondrial protein Miro and kinesin motors. In this study, we used gene silencing by targeted shRNAi and dominant negative approaches in conjunction with live imaging to investigate the contribution of endogenous TRAKs, TRAK1 and TRAK2, to the transport of mitochondria in axons of hippocampal pyramidal neurons. We report that both strategies resulted in impairing mitochondrial mobility in axonal processes. Differences were apparent in terms of the contribution of TRAK1 and TRAK2 to this transport because knockdown of TRAK1 but not TRAK2 impaired mitochondrial mobility, yet both TRAK1 and TRAK2 were shown to rescue transport impaired by TRAK1 gene knock-out. Thus, we demonstrate for the first time the pivotal contribution of the endogenous TRAK family of kinesin adaptors to the regulation of mitochondrial mobility.     

DISC1-dependent Regulation of Mitochondrial Dynamics Controls the Morphogenesis of Complex Neuronal Dendrites

The DISC1 protein is implicated in major mental illnesses including schizophrenia, depression, bipolar disorder, and autism. Aberrant mitochondrial dynamics are also associated with major mental illness. DISC1 plays a role in mitochondrial transport in neuronal axons, but its effects in dendrites have yet to be studied. Further, the mechanisms of this regulation and its role in neuronal development and brain function are poorly understood. Here we have demonstrated that DISC1 couples to the mitochondrial transport and fusion machinery via interaction with the outer mitochondrial membrane GTPase proteins Miro1 and Miro2, the TRAK1 and TRAK2 mitochondrial trafficking adaptors, and the mitochondrial fusion proteins (mitofusins). Using live cell imaging, we show that disruption of the DISC1-Miro-TRAK complex inhibits mitochondrial transport in neurons. We also show that the fusion protein generated from the originally described DISC1 translocation (DISC1-Boymaw) localizes to the mitochondria, where it similarly disrupts mitochondrial dynamics. We also show by super resolution microscopy that DISC1 is localized to endoplasmic reticulum contact sites and that the DISC1-Boymaw fusion protein decreases the endoplasmic reticulum-mitochondria contact area. Moreover, disruption of mitochondrial dynamics by targeting the DISC1-Miro-TRAK complex or upon expression of the DISC1-Boymaw fusion protein impairs the correct development of neuronal dendrites. Thus, DISC1 acts as an important regulator of mitochondrial dynamics in both axons and dendrites to mediate the transport, fusion, and cross-talk of these organelles, and pathological DISC1 isoforms disrupt this critical function leading to abnormal neuronal development.     

The ERMES complex and ER-mitochondria connections

Cellular organelles need to communicate in order to co-ordinate homoeostasis of the compartmentalized eukaryotic cell. Such communication involves the formation of membrane contact sites between adjacent organelles, allowing privileged exchange of metabolites and information. Using a synthetic protein designed to artificially tether the ER (endoplasmic reticulum) to mitochondria, we have discovered a yeast protein complex naturally involved in establishing and maintaining contact sites between these two organelles. This protein complex is physiologically involved in a plethora of mitochondrial processes, suggesting that ER-mitochondria connections play a central co-ordinating role in the regulation of mitochondrial biology. Recent biochemical characterization of this protein complex led to the discovery that GTPases of the Miro family are part of ER-mitochondria connections. The yeast Miro GTPase Gem1 localizes to ER-mitochondria interface and influences the size and distribution of mitochondria. Thus Miro GTPases may serve as regulators of the ER-mitochondria connection.     

PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility

Cells keep their energy balance and avoid oxidative stress by regulating mitochondrial movement, distribution, and clearance. We report here that two Parkinson's disease proteins, the Ser/Thr kinase PINK1 and ubiquitin ligase Parkin, participate in this regulation by arresting mitochondrial movement. PINK1 phosphorylates Miro, a component of the primary motor/adaptor complex that anchors kinesin to the mitochondrial surface. The phosphorylation of Miro activates proteasomal degradation of Miro in a Parkin-dependent manner. Removal of Miro from the mitochondrion also detaches kinesin from its surface. By preventing mitochondrial movement, the PINK1/Parkin pathway may quarantine damaged mitochondria prior to their clearance. PINK1 has been shown to act upstream of Parkin, but the mechanism corresponding to this relationship has not been known. We propose that PINK1 phosphorylation of substrates triggers the subsequent action of Parkin and the proteasome.     

Parkinson's disease-associated kinase PINK1 regulates Miro protein level and axonal transport of mitochondria

Mutations in Pten-induced kinase 1 (PINK1) are linked to early-onset familial Parkinson's disease (FPD). PINK1 has previously been implicated in mitochondrial fission/fusion dynamics, quality control, and electron transport chain function. However, it is not clear how these processes are interconnected and whether they are sufficient to explain all aspects of PINK1 pathogenesis. Here we show that PINK1 also controls mitochondrial motility. In Drosophila, downregulation of dMiro or other components of the mitochondrial transport machinery rescued dPINK1 mutant phenotypes in the muscle and dopaminergic (DA) neurons, whereas dMiro overexpression alone caused DA neuron loss. dMiro protein level was increased in dPINK1 mutant but decreased in dPINK1 or dParkin overexpression conditions. In Drosophila larval motor neurons, overexpression of dPINK1 inhibited axonal mitochondria transport in both anterograde and retrograde directions, whereas dPINK1 knockdown promoted anterograde transport. In HeLa cells, overexpressed hPINK1 worked together with hParkin, another FPD gene, to regulate the ubiquitination and degradation of hMiro1 and hMiro2, apparently in a Ser-156 phosphorylation-independent manner. Also in HeLa cells, loss of hMiro promoted the perinuclear clustering of mitochondria and facilitated autophagy of damaged mitochondria, effects previously associated with activation of the PINK1/Parkin pathway. These newly identified functions of PINK1/Parkin and Miro in mitochondrial transport and mitophagy contribute to our understanding of the complex interplays in mitochondrial quality control that are critically involved in PD pathogenesis, and they may explain the peripheral neuropathy symptoms seen in some PD patients carrying particular PINK1 or Parkin mutations. Moreover, the different effects of loss of PINK1 function on Miro protein level in Drosophila and mouse cells may offer one explanation of the distinct phenotypic manifestations of PINK1 mutants in these two species.     

Axonal transport of mitochondria to synapses depends on milton,a novel Drosophila protein

A protein required to localize mitochondria to Drosophila nerve terminals has been identified genetically. Photoreceptors mutant for milton show aberrant synaptic transmission despite normal phototransduction. Without Milton, synaptic terminals and axons lack mitochondria, although mitochondria are numerous in neuronal cell bodies. In contrast, synaptic vesicles continue to be transported to and concentrated at synapses. Milton protein is associated with mitochondria and is present primarily in axons and synapses. A likely explanation of the apparent trafficking defect is offered by the coimmunoprecipitation of Milton and kinesin heavy chain. Transfected into HEK293T cells, Milton induces a redistribution of mitochondria within the cell. We propose that Milton is a mitochondria-associated protein required for kinesin-mediated transport of mitochondria to nerve terminals.     

Miro: A molecular switch at the center of mitochondrial regulation

The orchestration of mitochondria within the cell represents a critical aspect of cell biology. At the center of this process is the outer mitochondrial membrane protein, Miro. Miro coordinates diverse cellular processes by regulating connections between organelles and the cytoskeleton that range from mediating contacts between the endoplasmic reticulum and mitochondria to the regulation of both actin and microtubule motor proteins. Recently, a number of cell biological, biochemical, and protein structure studies have helped to characterize the myriad roles played by Miro. In addition to answering questions regarding Miro's function, these studies have opened the door to new avenues in the study of Miro in the cell. This review will focus on summarizing recent findings for Miro's structure, function, and activity while highlighting key questions that remain unanswered.     

Loss of neuronal Miro1 disrupts mitophagy and induces hyperactivation of the integrated stress response

Clearance of mitochondria following damage is critical for neuronal homeostasis. Here, we investigate the role of Miro proteins in mitochondrial turnover by the PINK1/Parkin mitochondrial quality control system in vitro and in vivo. We find that upon mitochondrial damage, Miro is promiscuously ubiquitinated on multiple lysine residues. Genetic deletion of Miro or block of Miro1 ubiquitination and subsequent degradation lead to delayed translocation of the E3 ubiquitin ligase Parkin onto damaged mitochondria and reduced mitochondrial clearance in both fibroblasts and cultured neurons. Disrupted mitophagy in vivo, upon post‐natal knockout of Miro1 in hippocampus and cortex, leads to a dramatic increase in mitofusin levels, the appearance of enlarged and hyperfused mitochondria and hyperactivation of the integrated stress response (ISR). Altogether, our results provide new insights into the central role of Miro1 in the regulation of mitochondrial homeostasis and further implicate Miro1 dysfunction in the pathogenesis of human neurodegenerative disease.     

Understanding Miro GTPases: Implications in the Treatment of Neurodegenerative Disorders

The Miro GTPases represent an unusual subgroup of the Ras superfamily and have recently emerged as important mediators of mitochondrial dynamics and for maintaining neuronal health. It is now well-established that these enzymes act as essential components of a Ca²⁺-sensitive motor complex, facilitating the transport of mitochondria along microtubules in several cell types, including dopaminergic neurons. The Miros appear to be critical for both anterograde and retrograde mitochondrial transport in axons and dendrites, both of which are considered essential for neuronal health. Furthermore, the Miros may be significantly involved in the development of several serious pathological processes, including the development of neurodegenerative and psychiatric disorders. In this review, we discuss the molecular structure and known mitochondrial functions of the Miro GTPases in humans and other organisms, in the context of neurodegenerative disease. Finally, we consider the potential human Miros hold as novel therapeutic targets for the treatment of such disease.     

Alphaherpesvirus infection disrupts mitochondrial transport in neurons

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.     

Disruption of the gene for Hsp30, an alpha-crystallin-related heat shock protein of Neurospora crassa, causes defects in import of proteins into mitochondria

The gene for Hsp30, the only known alpha-crystallin-related heat shock protein of Neurospora crassa, was disrupted by repeat-induced point mutagenesis, leading to loss of cell survival at high temperature. Hsp30, which is not synthesized at 30 degrees C, associates reversibly with the mitochondria at high temperature (45 degrees C). In this study, we found that import of selected proteins into internal compartments of mitochondria, following their synthesis in the cytosol, was severely impaired at high temperature in a strain mutant in Hsp30. After 70 min of cell incubation at 45 degrees C, most matrix, inner membrane, and intermembrane-space proteins tested were reduced in import by about 50-70% in the mutant, as compared to wild-type cells. In contrast, assembly of selected proteins into the outer mitochondrial membrane was not reduced, except for one component of the preprotein translocase complex of the mitochondrial outer membrane. Three proteins of this complex co-immunoprecipitated with Hsp30 of wild-type cells incubated at 45 degrees C. We propose that Hsp30 interacts with the preprotein translocase of the mitochondrial outer membrane and that it chaperones the activity of one or more components of this translocase complex at high temperature.     

Brain-derived Neurotrophic Factor (BDNF)-induced Mitochondrial Motility Arrest and Presynaptic Docking Contribute to BDNF-enhanced Synaptic Transmission

Appropriate mitochondrial transport and distribution are essential for neurons because of the high energy and Ca²⁺ buffering requirements at synapses. Brain-derived neurotrophic factor (BDNF) plays an essential role in regulating synaptic transmission and plasticity. However, whether and how BDNF can regulate mitochondrial transport and distribution are still unclear. Here, we find that in cultured hippocampal neurons, application of BDNF for 15 min decreased the percentage of moving mitochondria in axons, a process dependent on the activation of the TrkB receptor and its downstream PI3K and phospholipase-Cγ signaling pathways. Moreover, the BDNF-induced mitochondrial stopping requires the activation of transient receptor potential canonical 3 and 6 (TRPC3 and TRPC6) channels and elevated intracellular Ca²⁺ levels. The Ca²⁺ sensor Miro1 plays an important role in this process. Finally, the BDNF-induced mitochondrial stopping leads to the accumulation of more mitochondria at presynaptic sites. Mutant Miro1 lacking the ability to bind Ca²⁺ prevents BDNF-induced mitochondrial presynaptic accumulation and synaptic transmission, suggesting that Miro1-mediated mitochondrial motility is involved in BDNF-induced mitochondrial presynaptic docking and neurotransmission. Together, these data suggest that mitochondrial transport and distribution play essential roles in BDNF-mediated synaptic transmission.