EMB2473/MIRO1, an Arabidopsis Miro GTPase, is required for embryogenesis and influences mitochondrial morphology in pollen

The regulation of mitochondrial biogenesis, subcellular distribution, morphology, and metabolism are essential for all aspects of plant growth and development. However, the molecular mechanisms involved are still unclear. Here, we describe an analysis of the three Arabidopsis thaliana orthologs of the evolutionarily conserved Miro GTPases. Two of the genes, MIRO1 and MIRO2, are transcribed ubiquitously throughout the plant tissues, and their gene products localize to mitochondria via their C-terminal transmembrane domains. While insertional mutations in the MIRO2 gene do not have any visible impact on plant development, an insertional mutation in the MIRO1 gene is lethal during embryogenesis at the zygote to four-terminal-cell embryo stage. It also substantially impairs pollen germination and tube growth. Laser confocal and transmission electron microscopy revealed that the miro1 mutant pollen exhibits abnormally enlarged or tube-like mitochondrial morphology, leading to the disruption of continuous streaming of mitochondria in the growing pollen tube. Our findings suggest that mitochondrial morphology is influenced by MIRO1 and plays a vital role during embryogenesis and pollen tube growth.     

Arabidopsis thaliana MIRO1 and MIRO2 GTPases are unequally redundant in pollen tube growth and fusion of polar nuclei during female gametogenesis

MIRO GTPases have evolved to regulate mitochondrial trafficking and morphology in eukaryotic organisms. A previous study showed that T-DNA insertion in the Arabidopsis MIRO1 gene is lethal during embryogenesis and affects pollen tube growth and mitochondrial morphology in pollen, whereas T-DNA insertion in MIRO2 does not affect plant development visibly. Phylogenetic analysis of MIRO from plants revealed that MIRO 1 and 2 orthologs in dicots cluster in two separate groups due to a gene/genome duplication event, suggesting that functional redundancy may exists between the two MIRO genes. To investigate this possibility, we generated miro1(+/-)/miro2-2(-/-) plants. Compared to miro1(+/-) plants, the miro1(+/-)/miro2-2(-/-) plants showed increased segregation distortion. miro1(+/-)/miro2-2(-/-) siliques contained less aborted seeds, but more than 3 times the number of undeveloped ovules. In addition, reciprocal crosses showed that co-transmission through the male gametes was nearly absent, whereas co-transmission through the female gametes was severely reduced in miro1(+/-)/miro2-2(-/-) plants. Further investigations revealed that loss of MIRO2 (miro2(-/-)) function in the miro1(+/-) background enhanced pollen tube growth defects. In developing miro1(+/-)/miro2(-/-) embryo sacs, fusion of polar nuclei was further delayed or impaired compared to miro1 plants. This phenotype has not been reported previously for miro1 plants and coincides with studies showing that defects in some mitochondria-targeted genes results in the same phenotype. Our observations show that loss of function in MIRO2 in a miro1(+/-) background enhances the miro1(+/-) phenotype significantly, even though miro2(-/-) plants alone does not display any phenotypes. Based on these findings, we conclude that MIRO1 and MIRO2 are unequally redundant and that a proportion of the miro1(+/-)/miro2(-/-) plants haploid gametes displays the complete null phenotype of MIRO GTPase function at key developmental stages.     

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.     

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.     

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.     

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.     

MitoTimer probe reveals the impact of autophagy,fusion, and motility on subcellular distribution of young and old mitochondrial protein and on relative mitochondrial protein age

To study mitochondrial protein age dynamics, we targeted a time-sensitive fluorescent protein, MitoTimer, to the mitochondrial matrix. Mitochondrial age was revealed by the integrated portions of young (green) and old (red) MitoTimer protein. Mitochondrial protein age was dependent on turnover rates as pulsed synthesis, decreased import, or autophagic inhibition all increased the proportion of aged MitoTimer protein. Mitochondrial fusion promotes the distribution of young mitochondrial protein across the mitochondrial network as cells lacking essential fusion genes Mfn1 and Mfn2 displayed increased heterogeneity in mitochondrial protein age. Experiments in hippocampal neurons illustrate that the distribution of older and younger mitochondrial protein within the cell is determined by subcellular spatial organization and compartmentalization of mitochondria into neurites and soma. This effect was altered by overexpression of mitochondrial transport protein, RHOT1/MIRO1. Collectively our data show that distribution of young and old protein in the mitochondrial network is dependent on turnover, fusion, and transport.     

Structure-function analysis of the yeast mitochondrial Rho GTPase, Gem1p: implications for mitochondrial inheritance

Mitochondria undergo continuous cycles of homotypic fusion and fission, which play an important role in controlling organelle morphology, copy number, and mitochondrial DNA maintenance. Because mitochondria cannot be generated de novo, the motility and distribution of these organelles are essential for their inheritance by daughter cells during division. Mitochondrial Rho (Miro) GTPases are outer mitochondrial membrane proteins with two GTPase domains and two EF-hand motifs, which act as receptors to regulate mitochondrial motility and inheritance. Here we report that although all of these domains are biochemically active, only the GTPase domains are required for the mitochondrial inheritance function of Gem1p (the yeast Miro ortholog). Mutations in either of the Gem1p GTPase domains completely abrogated mitochondrial inheritance, although the mutant proteins retained half the GTPase activity of the wild-type protein. Although mitochondrial inheritance was not dependent upon Ca(2+) binding by the two EF-hands of Gem1p, a functional N-terminal EF-hand I motif was critical for stable expression of Gem1p in vivo. Our results suggest that basic features of Miro protein function are conserved from yeast to humans, despite differences in the cellular machinery mediating mitochondrial distribution in these organisms.     

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.     

A Highlights from MBoC Selection: APC binds the Miro/Milton motor complex to stimulate transport of mitochondria to the plasma membrane

Mutations in adenomatous polyposis coli (APC) disrupt regulation of Wnt signaling, mitosis, and the cytoskeleton. We describe a new role for APC in the transport of mitochondria. Silencing of wild-type APC by small interfering RNA caused mitochondria to redistribute from the cell periphery to the perinuclear region. We identified novel APC interactions with the mitochondrial kinesin-motor complex Miro/Milton that were mediated by the APC C-terminus. Truncating mutations in APC abolished its ability to bind Miro/Milton and reduced formation of the Miro/Milton complex, correlating with disrupted mitochondrial distribution in colorectal cancer cells that could be recovered by reconstitution of wild-type APC. Using proximity ligation assays, we identified endogenous APC-Miro/Milton complexes at mitochondria, and live-cell imaging showed that loss of APC slowed the frequency of anterograde mitochondrial transport to the membrane. We propose that APC helps drive mitochondria to the membrane to supply energy for cellular processes such as directed cell migration, a process disrupted by cancer mutations.     

Miro1: New wheels for transferring mitochondria

Mesenchymal stem cells (MSC) are capable of protecting cells harboring mitochondrial damage. This protection is associated with the transfer of mitochondria through tunneling nanotubes (TNT) from MSC to the injured cells. In this issue of The EMBO Journal, the group of Anurag Agrawal shows that mitochondrial transfer is dependent on the levels of Miro1, a mitochondrial Rho‐GTPase that regulates intercellular mitochondrial movement. Miro1 is the first protein shown to accelerate mitochondrial transfer. Amplifying the mitochondrial transfer phenomenon may allow for the study of the mechanisms that regulate it and contribute to our understanding of its role in disease and aging.     

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.     

Mitochondrial morphology is dynamic and varied

The morphology of mitochondria is dynamic, often changing within a cell and from one cell type to the next. In the past few years, significant advances have been made in the study of mechanisms that help determine the morphologies of mitochondria and their intracellular distributions. It has become apparent that the distribution of mitochondria is determined by movement along the cytoskeleton, driven by molecular motors, and attachment to the cytoskeleton, using specific connector proteins. However, not all cells use the same cytoskeletal elements and motor proteins for mitochondrial movement and attachment. The shapes of mitochondria are also influenced by the extent of mitochondrial division and fusion. A number of proteins that affect mitochondrial division and fusion were recently discovered. Here, we review the proteins involved in the distribution and morphology of mitochondria and discuss how they may be physiologically regulated.     

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.     

Multiple pathways influence mitochondrial inheritance in budding yeast

Yeast mitochondria form a branched tubular network. Mitochondrial inheritance is tightly coupled with bud emergence, ensuring that daughter cells receive mitochondria from mother cells during division. Proteins reported to influence mitochondrial inheritance include the mitochondrial rho (Miro) GTPase Gem1p, Mmr1p, and Ypt11p. A synthetic genetic array (SGA) screen revealed interactions between gem1Delta and deletions of genes that affect mitochondrial function or inheritance, including mmr1Delta. Synthetic sickness of gem1Delta mmr1Delta double mutants correlated with defective mitochondrial inheritance by large buds. Additional studies demonstrated that GEM1, MMR1, and YPT11 each contribute to mitochondrial inheritance. Mitochondrial accumulation in buds caused by overexpression of either Mmr1p or Ypt11p did not depend on Gem1p, indicating these three proteins function independently. Physical linkage of mitochondria with the endoplasmic reticulum (ER) has led to speculation that distribution of these two organelles is coordinated. We show that yeast mitochondrial inheritance is not required for inheritance or spreading of cortical ER in the bud. Moreover, Ypt11p overexpression, but not Mmr1p overexpression, caused ER accumulation in the bud, revealing a potential role for Ypt11p in ER distribution. This study demonstrates that multiple pathways influence mitochondrial inheritance in yeast and that Miro GTPases have conserved roles in mitochondrial distribution.     

Late embryo-defective mutants of Arabidopsis

The embryo-defective (emb) mutants of Arabidopsis constitute a large and diverse group of mutants disrupted in a broad range of embryonic processes, including morphogonesis, cell differentiation, and maturation programs. This report describes a subset of these mutants, the late embryo defectives, which develop beyond the globular stage of embryogenesis but fail to complete normal morphogenesis. A representative sample of 12 late mutants was chosen for this study, patterns of morphogenesis were characterized, the germination potential of mutant seeds was investigated, and additional mutant alleles within the collection were identified. Morphological defects in mutant embryos became apparent during the heart stage of development, when embryos normally begin the rapid cell division and expansion required for the completion of morphogenesis. Despite their morphological abnormalities, mutant embryos often germinated from dry seed, demonstrating that genetic programs required for the establishment of desiccation tolerance remained intact. Mutant seedlings displayed a wide range of developmental abnormalities, including altered morphology, lack of pigmentation, dwarfism, and disorganized vegetative growth. One late mutant was found to be allelic to an early embryo defective that arrests at the globular stage. These results suggest that a number of late EMB genes encode basic cellular and metabolic functions needed for cell division, enlargement, and embryonic growth. The rapid growth and metabolic changes that occur at the heart stage may present a barrier to normal development in the late mutants, resulting in altered embryo morphology and other developmental defects. It is proposed that many Arabidopsis mutants with abnormal embryo and seedling morphology are not defective in the regulation of pattern formation or morphogenesis, but rather in fundamental physiological and cellular processes required for the completion of normal growth and development. © 1995 Wiley-Liss, Inc.     

An increase in the intracellular pH of fertilized eggs of Xenopus laevis is associated with inhibition of protein and DNA syntheses and followed by an arrest of embryonic development

In many systems, events participating in cell division are controlled by intracellular pH (pH_i). In Xenopus eggs, fertilization is accompanied by an increase in pH_i which occurs concomitantly with an increase in protein synthesis and a reinitiation of DNA synthesis, leading the embryo to cell division. In this paper, we have shown that increasing pH_i of fertilized eggs from 7.8 to 8.2 by using weak bases produced an arrest in embryonic development. Such a change in pH_i was accompanied by a severe inhibition of both protein and DNA syntheses. In order to discriminate between a direct effect of pH_i and a pH-independent effect of weak bases on these biosyntheses, the situation was studied in vitro. For this purpose, cytoplasmic extracts were used in which weak base addition did not produce any change in pH. Under these conditions, protein synthesis was not inhibited, suggesting that pH is probably one of the events implicated in the regulation of protein synthesis. On the other hand, DNA synthesis was inhibited by weak bases in vitro, without any change in pH intervening.     

Miro1‐dependent mitochondrial positioning drives the rescaling of presynaptic Ca2+ signals during homeostatic plasticity

Mitochondrial trafficking is influenced by neuronal activity, but it remains unclear how mitochondrial positioning influences neuronal transmission and plasticity. Here, we use live cell imaging with the genetically encoded presynaptically targeted Ca²⁺ indicator, SyGCaMP5, to address whether presynaptic Ca²⁺ responses are altered by mitochondria in synaptic terminals. We find that presynaptic Ca²⁺ signals, as well as neurotransmitter release, are significantly decreased in terminals containing mitochondria. Moreover, the localisation of mitochondria at presynaptic sites can be altered during long‐term activity changes, dependent on the Ca²⁺‐sensing function of the mitochondrial trafficking protein, Miro1. In addition, we find that Miro1‐mediated activity‐dependent synaptic repositioning of mitochondria allows neurons to homeostatically alter the strength of presynaptic Ca²⁺ signals in response to prolonged changes in neuronal activity. Our results support a model in which mitochondria are recruited to presynaptic terminals during periods of raised neuronal activity and are involved in rescaling synaptic signals during homeostatic plasticity.