Gene name - Mitochondrial Rho
Synonyms - dmiro
Cytological map position - 95D8--9
Function - signaling
Symbol - Miro
FlyBase ID: FBgn0039140
Genetic map position - 3R
Classification - Ras GTPase, calcium-binding EF-hand
Cellular location - mitochondrial membrane
|Recent literature||Babic, M., Russo, G. J., Wellington, A. J., Sangston, R. M., Gonzalez, M. and Zinsmaier, K. E. (2015). Miro's N-Terminal GTPase domain is required for transport of mitochondria into axons and dendrites. J Neurosci 35: 5754-5771. PubMed ID: 25855186
Mitochondria are dynamically transported in and out of neuronal processes to maintain neuronal excitability and synaptic function. In higher eukaryotes, the mitochondrial GTPase Miro binds Milton/TRAK adaptor proteins linking microtubule motors to mitochondria. This study shows that Drosophila Miro (dMiro), which has previously been shown to be required for kinesin-driven axonal transport, is also critically required for the dynein-driven distribution of mitochondria into dendrites. In addition, the loss-of-function mutations dMiroT25N and dMiroT460N were used to determine the significance of dMiro's N-terminal and C-terminal GTPase domains, respectively. Expression of dMiroT25N in the absence of endogenous dMiro caused premature lethality and arrested development at a pupal stage. dMiroT25N accumulated mitochondria in the soma of larval motor and sensory neurons, and prevented their kinesin-dependent and dynein-dependent distribution into axons and dendrites, respectively. dMiroT25N mutant mitochondria also were severely fragmented and exhibited reduced kinesin and dynein motility in axons. In contrast, dMiroT460N did not impair viability, mitochondrial size, or the distribution of mitochondria. However, dMiroT460N reduced dynein motility during retrograde mitochondrial transport in axons. Finally, this study showed that substitutions analogous to the constitutively active Ras-G12V mutation in dMiro's N-terminal and C-terminal GTPase domains cause neomorphic phenotypic effects that are likely unrelated to the normal function of each GTPase domain. Overall, this analysis indicates that dMiro's N-terminal GTPase domain is critically required for viability, mitochondrial size, and the distribution of mitochondria out of the neuronal soma regardless of the employed motor, likely by promoting the transition from a stationary to a motile state.
|Ding, L., Lei, Y., Han, Y., Li, Y., Ji, X. and Liu, L. (2016). Vimar is a novel regulator of mitochondrial fission through Miro. PLoS Genet 12: e1006359. PubMed ID: 27716788
As fundamental processes in mitochondrial dynamics, mitochondrial fusion, fission and transport are regulated by several core components, including Miro. As an atypical Rho-like small GTPase with high molecular mass, the exchange of GDP/GTP in Miro may require assistance from a guanine nucleotide exchange factor (GEF). However, the GEF for Miro has not been identified. While studying mitochondrial morphology in Drosophila, it was incidentally observed that the loss of vimar, a gene encoding an atypical GEF, enhanced mitochondrial fission under normal physiological conditions. Because Vimar could co-immunoprecipitate with Miro in vitro, it was speculated that Vimar might be the GEF of Miro. In support of this hypothesis, a loss-of-function (LOF) vimar mutant rescued mitochondrial enlargement induced by a gain-of-function (GOF) Miro transgene; whereas a GOF vimar transgene enhanced Miro function. In addition, vimar lost its effect under the expression of a constitutively GTP-bound or GDP-bound Miro mutant background. These results indicate a genetic dependence of vimar on Miro. Moreover, mitochondrial fission was found to play a functional role in high-calcium induced necrosis, and a LOF vimar mutant rescued the mitochondrial fission defect and cell death. This result can also be explained by vimar's function through Miro, because Miro's effect on mitochondrial morphology is altered upon binding with calcium. In addition, a PINK1 mutant, which induced mitochondrial enlargement and had been considered as a Drosophila model of Parkinson's disease (PD), caused fly muscle defects, and the loss of vimar could rescue these defects. Furthermore, it was found that the mammalian homolog of Vimar, RAP1GDS1, played a similar role in regulating mitochondrial morphology, suggesting a functional conservation of this GEF member. The Miro/Vimar complex may be a promising drug target for diseases in which mitochondrial fission and fusion are dysfunctional.
|Melkov, A., Baskar, R., Alcalay, Y. and Abdu, U. (2016). New mode of mitochondrial transport and polarized sorting regulation by Dynein, Milton and Miro. Development [Epub ahead of print]. PubMed ID: 27707795
Intrinsic cell microtubule (MT) polarity, together with molecular motors and adaptor proteins, determines mitochondrial polarized targeting and MT-dependent transport. In polarized cells, such as neurons, mitochondrialmobility and transport require the regulation of kinesin and dynein by two adaptor proteins, Milton and Miro. Recent, studies have found that Dynein heavy chain 64C (Dhc64C) is the primary motor protein for both anterograde and retrograde transport of mitochondria in the Drosophila bristle. This study revealed that a molecular lesion in the Dhc64C allele that reduced bristle mitochondrial velocity generated a variant that acts as a "slow" dynein in a MT gliding assay, indicative of dynein directly regulating mitochondrial transport. It was also shown that in milton RNAi flies, mitochondrial flux into the bristle shaft but not velocity was significantly reduced. Surprisingly, mitochondria retrograde flux but not net velocity was significantly decreased in miro RNAi flies. This study thus revealed a new mode of mitochondrial polarized sorting in polarized cell growth, whereby bi-directional mitochondrial transport undertaken exclusively by dynein is regulated by Milton in anterograde direction and by a Miro-dependent switch to retrograde direction.
EMS-induced mutations have been identified in Drosophila Miro, an atypical mitochondrial GTPase that is orthologous to human Miro (hMiro). Mutant dmiro animals exhibit defects in locomotion and die prematurely. Mitochondria in Miro 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 impaired only during prolonged stimulation. Neuronal, but not muscular, expression of Miro in Miro mutants restores viability, the 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 Miro function causes an abnormal accumulation of mitochondria in distal synaptic boutons of NMJs. Together, these findings suggest that Miro is required for controlling anterograde transport of mitochondria and their proper distribution within nerve terminals (Guo, 2005).
Mitochondria are critical for aerobic respiration, Ca2+ homeostasis, and apoptosis. The activity and subcellular distribution of mitochondria are not static, but adaptable to physiological stresses and changes in the metabolic demands of the cell. Control of mitochondrial distribution is believed to be especially important for neurons because of their high metabolic demands and their complex polar morphology (e.g., axons, dendrites, and synapses). Neuronal mitochondria are enriched in regions of intense energy consumption, including mobile growth cones, nodes of Ranvier, and synaptic terminals. For example, synaptic terminals may consume up to 10% of the total energy required for neuronal signaling. Consistently, loss of mitochondria from photoreceptor terminals is associated with blindness and a failure of synaptic transmission in the Drosophila mutant milton (Stowers, 2002). In cultured hippocampal neurons, the number of dendritic mitochondria correlates with the number and plasticity of dendritic spines and synapses. In addition, mitochondrial transport responds specifically to growth cone activity and nerve growth factor signaling. Together, these studies underscore the functional significance of controlling the subcellular targeting of mitochondria (Guo, 2005).
The molecular mechanisms that control the subcellular distribution of mitochondria involve long-distance transport along microtubules (MTs), which provide polar tracks for plus end-directed kinesin and minus end-directed dynein motor proteins. Mitochondria, like vesicles, display bidirectional motion where the cargo stops, starts, and often changes direction (Welte, 2004; Vale, 2003; Hollenbeck, 1996; Hollenbeck, 2005). Assuming that mitochondria are simultaneously attached to two opposing motors (De Vos, 2003), then net movement in one direction may be determined by the motor with the overall highest activity or, alternatively, only one of the motors may be engaged with the MT track (Welte, 2004). However, the molecular mechanisms that achieve specificity, directionality, and temporal control of mitochondrial transport in response to intracellular signals remain poorly understood. A better understanding is highly desirable, as vulnerabilities of transport systems to genetic and/or environmental insults often result in human neurological or neurodegenerative diseases (Guo, 2005 and references therein).
The mitochondrial Rho-GTPase (Miro) protein family may have the potential to link cellular signaling pathways with mitochondrial dynamics and function. Orthologs of human Miro (hMiro1 and 2) are all characterized by the presence of two different GTPase domains and two Ca2+ binding EF hand domains (Frederick, 2004; Fransson, 2003). Overexpression of constitutively active human Miro in COS7 cells causes perinuclear mitochondrial aggregates and increased apoptosis (Fransson, 2003). Yeast cells lacking Miro (Gem1p) reveal a collapse of the tubular mitochondrial network that is not caused by defects in mitochondrial fission and/or fusion. Genetic studies further indicate that both GTPase domains and both EF-hand motifs are required for Gem1p function (Guo, 2005).
A genetic screen was carried out for mutations that affect synaptic structure and function in Drosophila. From this screen, lethal mutations were identified in Drosophila Miro (Miro). Molecular and genetic analyses suggest that Miro is required for anterograde axonal transport of mitochondria and their proper subcellular distribution. Although Miro mutant motor terminals are structurally deformed and chronically lack mitochondria, they can sustain neurotransmitter release at basic levels, but fatigue during high-frequency stimulation. While presynaptic Ca2+ resting levels are elevated, abnormal accumulations of Ca2+ occur only during prolonged periods of repetitive stimulation (Guo, 2005).
Thus Miro is essential for the proper distribution of mitochondria into dendrites and axon terminals. Although the true cause for the slim body, the smaller muscle size, the progressive deterioration of locomotion, and the premature death of Miro mutant larvae remains to be established, it is remarkable that all of these deficiencies have an exclusively neuronal origin, since they are rescued by neuronal expression of normal Miro (Guo, 2005).
Motor nerve terminals of Miro null mutants lack mitochondria, but contain relatively undisturbed numbers of synaptic vesicles. Instead of being transported, mitochondria accumulate like 'strings of pearls' in neuronal somata, which indicates a traffic jam of mitochondria that are connected to microtubles but cannot be transported into axons and dendrites. This defect is unlikely to be caused by a structural or functional deficit of mitochondria, since they exhibit neither an ultrastructural defect nor a reduced mitochondrial membrane potential. The altered microtuble organization is also unlikely to cause the defect. Since both mitochondria and vesicles employ kinesin motors for anterograde transport, one would expect that a defect of microtubles affects mitochondrial and vesicular transport in qualitatively and quantitatively similar ways. However, neither the loss nor the gain of Miro activity affects transport mechanisms of these organelles in the same way. Hence, it is unlikely that the altered organization of microtubles is the primary cause of the defect in mitochondrial transport (Guo, 2005).
The mitochondrial accumulations of Miro mutants are similar to the perinuclear accumulations of microtuble-associated mitochondria in mouse mutants of the mitochondria-associated kinesin heavy chain Kif5B (Tanaka, 1998). Mutations in Drosophila Milton, a potential adaptor protein that links mitochondria to kinesin motors, also causes mitochondrial accumulations in photoreceptor somata and a loss of mitochondria at photoreceptor terminals (Gorska-Andrzejak, 2003; Stowers, 2002). Accordingly, the mitochondrial clusters in Miro mutants are consistent with an impairment of the anterograde transport machinery (Guo, 2005).
The effects of Miro overexpression further support a role in regulating mitochondrial transport. While expression of normal Miro protein in Miro mutants restored the transport of mitochondria out of somata into axons, it also caused an abnormal accumulation of mitochondria in terminal boutons of NMJs. This new defect is apparently induced by a gain of activity since similar alterations were also observed upon overexpression of Miro in otherwise wild-type flies. Hence, loss and gain of Miro activity consistently alter the subcellular distribution of mitochondria in neurons in opposite ways: while loss of Miro arrests mitochondrial transport in cell bodies, gain of Miro activity accumulates mitochondria at terminal synaptic boutons of motor axons. Three possibilities may explain the gain-of-function phenotype: excessive anterograde movement, a failure to terminate anterograde movement, or a failure in identifying appropriate subcellular target sites. Although this study cannot distinguish among these possibilities, the opposing effects induced by loss and gain of Miro activity consistently suggest that Miro is required for anterograde transport of mitochondria to ensure a normal subcellular distribution. Such a role is consistent with the suggestion that small GTPases, but not heterotrimeric G proteins, regulate organelle transport along axonal microtubles (Guo, 2005).
Assuming that Miro proteins, like other small GTPases, provide 'signaling nodes' that integrate signals to coordinate multiple downstream events, Miro proteins may provide an interface between cellular signaling pathways and mitochondrial transport to control the subcellular distribution of mitochondria. Such a signal-integrating role is supported by a structural analysis of yeast Miro, showing that both GTPase domains and both Ca2+ binding EF-hands are required for Gem1p function (Frederick, 2004). Accordingly, guanine nucleotide exchange and hydrolysis factors or Ca2+ binding could potentially modulate Miro activity and mitochondrial mobility at 'turnaround' or stationary 'target zones'. However, further work will be required to resolve how mitochondrial transport might be mediated by an independent or cooperative action of Miro GTPase and EF-hand domains (Guo, 2005).
The subcellular distribution of mitochondria in neurons is assumed to be important for neuronal physiology, but direct evidence is scarce. This study has uncovered a connection between presynaptic mitochondria and the structure of NMJs. The loss of Miro activity results in the loss of presynaptic mitochondria, an increased number of synaptic boutons, and an altered bouton structure, suggesting that presynaptic mitochondria and/or mitochondrial proteins are important for structurally organizing NMJs. However, presynaptic mitochondria at NMJs are not required for the formation of new synaptic boutons, which contrasts with the role that has been suggested (Li, 2004) for mitochondria in dendritic spine formation (Guo, 2005).
Li (2004) manipulated the GTPases Drp1 (dynamin-related protein 1) and Opa1 (optic atrophy), both of which alter the morphology and distribution of mitochondria by controlling mitochondrial fission and fusion. Manipulations that decreased the number of mitochondria in dendrites of cultured hippocampal cells reduced the number of synapses and dendritic spines. Reciprocally, increasing dendritic mitochondrial content or activity caused an increase in the number of synapses and dendritic spines, suggesting that dendritic mitochondria are rate limiting for the support of synapses (Li, 2004). The contrasting lack of any correlation between synapse number and the number of presynaptic mitochondria at Miro mutant NMJs may indicate different roles of pre- and postsynaptic mitochondria or differences between NMJs and central synapses. There also may be a critical difference between a complete absence and a reduction of mitochondria, since reduced numbers of mitochondria in hippocampal dendrites do not affect dendritic patterns (Li, 2004), while the loss of mitochondria at Miro mutant NMJs caused significant presynaptic structural changes. Consistently, loss of Drp1 function in Drosophila neither alters the structure nor increases the number of synaptic boutons at NMJs (see Verstreken, 2005), although the number of mitochondria is much reduced (Guo, 2005).
The abnormal structure of synaptic boutons at Miro mutant NMJs may be linked to the abnormal organization of presynaptic microtubles, as indicated by the loss of microtuble loops and bundles. The cause of the abnormal presynaptic microtuble organization at Miro mutant NMJs remains unknown, but chronic ATP depletion may be excluded because missing microtubles were also observed in Miro mutant muscles in which the general prevalence of abnormally clustered mitochondria is unlikely to result in areas of ATP depletion (Guo, 2005).
Motor nerve terminals of Miro mutants, with their chronic absence of mitochondria, provide interesting insights into the role of presynaptic mitochondria in synaptic function. The high energy costs of synaptic transmission, arising mostly from the ATP dependence of synaptic vesicle exo- and endocytosis, ion pumps, transporters, and transmitter metabolism, suggest that synaptic function requires the continuous presence of mitochondria. Although the presynaptic defects of Miro mutant nerve terminals support this notion, it is still surprising how well these synapses can adjust to the chronic lack of mitochondria. Since larval Miro motor terminals can maintain basic synaptic function for at least several days, the question arises of how these terminals are supplied with ATP. Potentially, diffusion of ATP from the cell body through the motor axon, together with local glycolysis, could substitute for oxidative ATP synthesis by presynaptic mitochondria (Guo, 2005).
Motor nerve terminals that chronically lack mitochondria provide a unique opportunity for studying the role of mitochondrial Ca2+ uptake in transmitter release. Acute pharmacological inactivation of mitochondria at NMJs of frog, lizard, and mouse suggest that mitochondrial Ca2+ uptake critically limits the accumulation of presynaptic Ca2+ during repetitive stimulation, thereby preventing desynchronization of evoked release (David, 2003 and Talbot, 2003). Recordings of presynaptic Ca2+ and transmitter release from Miro NMJs reveal remarkable differences between nerve terminals that acutely or chronically lack mitochondrial function. Chronic absence of mitochondria causes nanomolar increases in presynaptic Ca2+ levels during comparable repetitive stimulation, but not micromolar increases as reported for acute manipulations (David, 2003, Talbot, 2003, Suzuki, 2002; David, 1999; Tang, 1997). Since mitochondrial Ca2+ uptake occurs at Drosophila motor nerve terminals, it is suggested that mitochondrial Ca2+ uptake for Ca2+ homeostasis at these nerve terminals is either not required or easily compensated by other mechanisms. Assuming that the latter occurs, then it is surprising how powerful and effective these compensatory mechanisms are. A likely candidate for compensation is the endoplasmic reticulum, which interacts with mitochondria, exchanges Ca2+ with mitochondria, and can act as a Ca2+ sink (Rizzuto, 2000). Alternatively, Na+/Ca2+ exchange or membrane Ca2+ ATPase activities may be altered (Guo, 2005).
This study did not reveal a large, activity-dependent increase in presynaptic Ca2+ levels that correlated with the desynchronization of transmitter release, which contrasts with other studies in which mitochondria were acutely inactivated (David, 2003; Talbot, 2003). Consequently, the desynchronization of transmitter release in Miro mutants may be due either to the abnormal synaptic structure or the lack of mitochondrial ATP production impairing mobilization of synaptic vesicles in the reserve pool (Verstreken, 2005). In conclusion, the results reveal that the chronic loss of presynaptic mitochondria at Drosophila NMJs has severe consequences for presynaptic structure and neurotransmitter release, but unexpectedly mild consequences for presynaptic Ca2+ homeostasis (Guo, 2005).
The Miro gene spans ~3.5 kb of genomic DNA, consists of four exons, and expresses at least three mRNA transcripts derived by alternative splicing of exon 3. The predicted Miro protein sequences are orthologous (51% identity; 330/645) to human Miro (Fransson, 2003). Miro proteins contain two different GTPase domains and two conserved EF-hand domains. The N-terminal GTPase domain of Miro is most closely related to Rho-GTPases, while the second GTPase domain is related more to Rab-GTPases. Like the human and yeast orthologs, Miro contains a conserved transmembrane (TM) domain at its C terminus, which in yeast (Frederick, 2004) tail-anchors Miro in the outer mitochondrial membrane such that the functional domains are exposed to the cytoplasm (Guo, 2005).
Cell signaling events elicit changes in mitochondrial shape and activity. However, few mitochondrial proteins that interact with signaling pathways have been identified. Candidates include the conserved mitochondrial Rho (Miro) family of proteins, which contain two GTPase domains flanking a pair of calcium-binding EF-hand motifs. Gem1p (yeast Miro; encoded by YAL048C) is a tail-anchored outer mitochondrial membrane protein. Cells lacking Gem1p contain collapsed, globular, or grape-like mitochondria. Gem1p is not an essential component of characterized pathways that regulate mitochondrial dynamics. Genetic studies indicate both GTPase domains and EF-hand motifs, which are exposed to the cytoplasm, are required for Gem1p function. Although overexpression of a mutant human Miro protein caused increased apoptotic activity in cultured cells, Gem1p is not required for pheromone-induced yeast cell death. Thus, Gem1p defines a novel mitochondrial morphology pathway which may integrate cell signaling events with mitochondrial dynamics (Frederick, 2004).
The human genomic sequencing effort has revealed the presence of a large number of Rho GTPases encoded by the human genome. Reported in this study is the characterization of a new family of Rho GTPases with atypical features. These proteins, which were called Miro-1 and Miro-2 (for mitochondrial Rho), have tandem GTP-binding domains separated by a linker region containing putative calcium-binding EF hand motifs. Genes encoding Miro-like proteins were found in several eukaryotic organisms from Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster to mammals, indicating that these genes evolved early in evolution. Immunolocalization experiments, in which transfected NIH3T3 and COS 7 cells were stained for ectopically expressed Miro as well as for the endogenous Miro-1 protein, showed that Miro was present in mitochondria. Interestingly, overexpression of a constitutively active mutant of Miro-1 (Miro-1/Val-13) induce an aggregation of the mitochondrial network and results in an increases apoptotic rate of the cells expressing activated Miro-1. These data indicate a novel role for Rho-like GTPases in mitochondrial homeostasis and apoptosis (Fransson, 2003).
date revised: 22 February 2005
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