To verify the predicted mitochondrial localization for Miro, the subcellular localization of EGFP-tagged Miro protein was examined in transiently transfected COS7 cells. Strong ectopic overexpression of Miro-EGFP disrupts the subcellular mitochondrial distribution and causes a loss of mitochondria, but does not grossly affect MTs. Many cells also showed signs of apoptotic cell death. To minimize these dominant effects on protein localization, for analysis only weakly transfected COS7 cells were used in which ectopically expressed Miro colocalizes with mitochondria that exhibit a relatively normal distribution. To confirm a mitochondrial localization in flies, neuronal cell cultures were generated from larval brains transgenically expressing V5-tagged Miro protein. Double immunolabeling showed that tagged Miro protein colocalizes with the mitochondrial protein Milton (Stowers, 2002) in neuronal cell bodies and in small punctae in neurites, which are reminiscent of mitochondria. Subcellular fractionation of Drosophila head extracts, using glycerol gradients, showed that Miro, Milton, and cytochrome c copurify in mitochondrial fractions. Together, these studies confirm that Miro is associated with mitochondria (Guo, 2005).
Genetic screens were employed to identify previously unknown genes required for axonal and synaptic function in Drosophila. To bypass the inefficiency of lethal F3-screening methods, advantage was taken of the EGUF/Hid method, which produces genetically mosaic flies in which only the eye is exclusively composed of cells homozygous for the mutation. Since 'blindness' is caused by a loss of either phototransduction, nerve excitation, or synaptic function, a screen was carried out for 'blind' flies by assaying the phototactic behavioral response of Drosophila, using the 'counter-current apparatus' (Benzer, 1967). Targeting chromosome 3R, ~13,000 EMS-mutagenized F1 flies were screened and 102 mutations were recovered that disrupt phototaxis, but do not grossly affect eye morphology. Genetic complementation analysis revealed a total of 53 lethal complementation groups, which were further characterized by their genetic map position and their synaptic defects at larval NMJs (Guo, 2005).
The lethal mutation B682 was selected for further analysis because it caused an unusual activity-dependent defect in synaptic transmission and a lack of mitochondria at nerve terminals. Deletion mapping limited the B682 locus to a region containing 17 mostly predicted genes at chromosomal position 95D7-11. An independent genetic screen had also identified four lethal EMS-induced alleles that mapped to 95D7-11 and caused defects in synaptic transmission (Babcock, 2003). Further genetic analysis revealed that B682 failed to complement the lethality of all four alleles, suggesting that all five alleles disrupt the same gene (Guo, 2005).
Identification of the mutated gene was facilitated by genetic fine mapping of the B682 mutation using site-specific male recombination. The data placed the B682 locus between two P insertion sites, containing three predicted genes. Further genetic analysis excluded one flanking gene, leaving only two viable candidates: CG5977, the fly homolog of human Spastin and CG5410, the fly ortholog of human Miro (Guo, 2005).
Sequencing of the spastin and Miro genes in all five alleles, using genomic DNA and RT-PCR-derived cDNA, revealed that four of the alleles (B682, sd23, sd26, and sd32) contained mutations in Drosophila miro (Miro), but not in the spastin gene. One allele, sd10, exhibited mutations in both genes and is molecularly a double mutant. The Miro and dspastin genes are transcribed in opposite directions, but are separated by only ~306 bp. To exclude effects of Miro mutations on spastin expression, spastin RNA expression was examined by RT-PCR. Only the predicted double mutant sd10 showed reduced spastin RNA expression. Together, the molecular and genetic data consistently suggest that the alleles B682, sd23, sd26, and sd32 are single gene mutations of the Miro gene. The premature truncation of Miro protein in B682 and sd32 within the first GTPase domain suggests that both alleles are presumably null mutations. Consistently, phenotypes of homozygous B682 and sd32 mutants are indistinguishable from those of transheterozygous combinations involving the deletion Df(3R)mbc-R (Guo, 2005).
Miro is an essential gene; all examined Miro alleles are recessive larval to early-pupal lethals. Homozygous Miro mutant larvae are slim and exhibit abnormal locomotion. Control larvae crawl steadily in a given direction, showing rhythmic muscle contractions that run over the entire body. In contrast, homozygous Miro larvae tend to wiggle on the spot and gain little distance. Posterior segments of Miro larvae appear to be excessively sluggish and sometimes even paralyzed, while anterior segments are often excessively bent, as if to compensate for failing muscle contractions in the more posterior segments. This abnormal crawling behavior indicates a progressive weakness of the larval body-wall musculature that originates at posterior segments and eventually leads to paralysis (Guo, 2005).
To test whether Miro mutations affect mitochondria, live mitochondria were imaged by visualizing mitochondria selectively in neurons or muscles, using a modified GFP that specifically labels mitochondria (MitoGFP). Neuronal expression of MitoGFP revealed an abnormal distribution of neuronal mitochondria in ventral ganglia of Miro mutant larvae. In contrast to controls, MitoGFP fluorescence in Miro mutants was enriched in neuronal somata, but reduced in the neuropil, reduced in segmental nerves close to the ganglia, and absent in more distal nerve regions. At larval NMJs, presynaptic MitoGFP fluorescence was, in general, not detectable; only rarely mutant NMJs showed one or two punctae of GFP fluorescence while almost all synaptic boutons of control NMJs were labeled. The absence of presynaptic mitochondria at larval NMJs of Miro mutants was confirmed by live dye staining using MitoTracker dyes and by an ultrastructural analysis. Together, these results demonstrate that Miro mutant mitochondria are retained in neuronal cell bodies and are not properly distributed into neuronal processes (Guo, 2005).
Muscle-specific expression of MitoGFP reveals a highly regular and stereotypical pattern of mitochondria in control muscles, which is reminiscent of the pattern revealed by actin-phalloidin stainings. While the distribution of actin is normal in Miro mutant muscles, the distribution of mitochondria is severely disrupted: mitochondria were found in small clusters that were distributed over the entire muscle. The abnormal mitochondrial clusters in mutant muscles were not associated with nuclei, which contrasts with the abnormal, perinuclear mitochondrial clusters in mutant neurons (Guo, 2005).
An ultrastructural analysis provided further insights into the nature of the mitochondrial accumulations in neuronal somata. Sections of proximal larval nerves showed reduced numbers of mitochondria in axons and in the processes of glial cells of Miro mutants in comparison to control larvae. Reduced numbers of mitochondria were also found in the neuropil of Miro mutant ventral ganglia. In contrast, neuronal somata of Miro mutants exhibited increased numbers of mitochondria, while control neuronal somata contained few mitochondria. Specifically, Miro mutant somata showed large, organized clusters of mitochondria, which were variable in size and close to Golgi apparati. Within these clusters, mitochondria were neatly lined up in parallel rows, like 'strings of pearls'. In many cases, the mitochondria within a string showed a similar orientation of their cristae. The occurrence of organized mitochondrial clusters in Miro mutants has two implications: (1) the 'string of pearls' appearance suggests that the clustered mitochondria are associated with the cytoskeleton, presumably with MTs, and (2) it suggests that a MT-associated 'roadblock' or impaired anterograde motor activity may arrest anterograde mitochondrial transport (Guo, 2005).
Structural and/or functional changes in mitochondria often cause a redistribution of mitochondria and could also account for the defect in mitochondrial transport of Miro mutants. However, EM analysis revealed no noticeable ultrastructural defects of mitochondria in Miro mutants. In addition, mitochondria in cultured neurons from both Miro mutant and control ganglia showed similar intensities of MitoTracker staining. Finally, labeling of mitochondria with JC-1 dye showed no abnormalities in the membrane potential of Miro mutant mitochondria. Hence, it is unlikely that structural or functional changes of mitochondria underlie the abnormal transport of mitochondria in Miro mutants (Guo, 2005).
To test whether the arrest of mitochondrial transport in Miro mutants correlates with a defect in vesicular transport, larval nerves were examined for the presence of abnormal accumulations of synaptic vesicles, as such accumulations are typically associated with mutations affecting axonal transport. Immunostainings of Miro mutant larval nerves revealed a significant accumulation of immunopositive punctae for the synaptic vesicle protein cysteine string protein (CSP) and synaptotagmin. However, quantification of synaptic vesicle staining at NMJs revealed no significant differences from controls, suggesting that the defect in vesicular transport is weak and does not limit vesicle supply. Similarly, immunostainings with mAb nc82, a presynaptic marker for synapses, also showed an accumulation of discrete nc82-positive punctae in Miro mutant nerves, while the number of synapses at NMJs was not reduced. Since nc82-positive punctae are very small and sparsely distributed in control nerves, the numerous large punctae in mutant nerves suggest an abnormal accumulation of transported cargo vesicles. While these data suggest that vesicular axonal transport is impaired in Miro mutants, these impairments are also qualitatively and quantitatively different from the defects in mitochondrial transport. Hence, it seems unlikely that both transport defects are caused by the impairment of a common mechanism (Guo, 2005).
Consistent with the smaller body size of Miro mutant larvae, the length of larval body-wall muscles was reduced in homozygous Miro mutants. However, the normalized length of the NMJ innervating the smaller muscle (NMJ/muscle ratio) of Miro mutants was generally increased to ~121% of that in controls. In addition, Miro mutant NMJs exhibit a general increase in the number of boutons to ~148% of that in controls after normalizing to the NMJ length, indicating synaptic overgrowth (Guo, 2005).
Close examination of Miro mutant synaptic boutons reveals an abnormal structure. The average volume of synaptic boutons is reduced, and the distance between individual boutons is shortened such that they were often aggregated in cauliflower-like clusters. Dextran-conjugated dye fillings or double immunostainings revealed individual compartments within these clusters, containing synaptic vesicles and synapses. An ultrastructural 3-D reconstruction from serial sections of synaptic boutons confirmed the smaller size of Miro boutons, their tendency to form densely packed clusters, and the absence of mitochondria. As already indicated by immunostainings, each compartment or bouton within a cluster contained its own set of synapses and synaptic vesicles (Guo, 2005).
To determine whether the altered bouton structure correlates with an abnormal microtubule cytoskeleton, presynaptic microtubules were examined, using anti-HRP and anti-acetylated tubulin double stainings. Control NMJs showed robust presynaptic microtubule bundles extending through the entire NMJ, and large synaptic boutons exhibited easily identifiable microtubule loops. In Miro mutants the number of microtubule loops was significantly reduced at all examined NMJs. In contrast to controls, presynaptic microtubule bundles at Miro mutant NMJs also often fail to extend into the last synaptic bouton of axonal branches. Hence, the altered presynaptic microtubule cytoskeleton of Miro mutant NMJs may contribute to the abnormal synaptic bouton structure (Guo, 2005).
The abnormal microtubule organization could be caused by the loss of Miro activity, the loss of other mitochondrial proteins, or by a depletion of ATP due to the loss of presynaptic mitochondria. To address this, microtubles were examined in Miro mutant muscles where a steep ATP gradient seems unlikely because mitochondrial clusters are uniformly distributed throughout the entire muscle. Microtubles surrounding cell nuclei appear more pronounced in Miro mutant muscles than in those of controls. However, quantification of microtubles surrounding individual nuclei showed no differences, while quantification of peripheral microtubles (arbitrarily defined as microtubles midway between neighboring nuclei) showed a significant reduction of microtuble filament density in Miro mutants, suggesting that microtuble organization in the muscle is altered under conditions in which ATP depletion is unlikely. In contrast, phalloidin staining of actin in muscles was normal in Miro mutants, indicating that Miro mutations selectively affect the microtuble, but not the actin cytoskeleton (Guo, 2005).
It seemed likely that mitochondrial transport requires Miro cell-autonomously. However, the structural defects of Miro nerve terminals may have been caused by the loss of pre- or postsynaptic Miro. To resolve this, normal Miro protein was selectively expressed in either neurons or muscles of Miro mutants, using two independent transgenes, which produced similar results. Remarkably, neuronal, but not muscular, expression of Miro rescues the lethality of Miro mutants, suggesting that Miro activity is essential only for neuronal, but not for non-neuronal, function. In addition, neuronal, but not muscular, expression of Miro restores normal larval body shape and muscle size. Neuronal expression does not rescue the abnormal mitochondrial distribution and/or microtuble organization in Miro mutant muscles. Hence, the smaller body and muscle size of Miro mutant larvae is not caused by muscle abnormalities, but by an abnormally functioning nervous system compromising the function of NMJs or neurosecretory terminals (Guo, 2005).
Neuronal expression of Miro in Miro mutants restores vesicular and mitochondrial transport in neurons such that mitochondria are again present at synaptic terminals of NMJs and in the neuropil of ventral ganglia. Presynaptic expression also restores normal synaptic bouton structure and reverses the absence of presynaptic microtuble loops at NMJs. However, postsynaptic expression of Miro in Miro mutant muscles neither reverses the defects in mitochondrial transport of the presynaptic neuron nor affects the abnormal structure of synaptic boutons and presynaptic microtubles. Together, these data suggest that Miro is required presynaptically to supply synaptic terminals with mitochondria and ensure normal structure of the NMJ (Guo, 2005).
While presynaptic expression of Miro restores mitochondrial transport to Miro mutant nerve terminals, it does not lead to a normal distribution of mitochondria; mitochondria accumulate abnormally in the most distal synaptic bouton of each terminal branch and are present in reduced numbers in most of the remaining boutons. The abnormal accumulation was also observed for DiOC2(5)-labeled mitochondria, confirming structural mitochondrial integrity. Overexpression of Miro in control neurons showed a similar, excessive accumulation of mitochondria in the terminal bouton of axonal branches, suggesting that this new phenotype is caused by a gain of Miro activity. This gain-of-function phenotype is not observed for synaptic and cargo vesicles since neither synaptic vesicles nor synapses were abnormally distributed. Hence, reducing or increasing Miro activity alters the subcellular distribution of mitochondria such that abnormal mitochondrial accumulations are switched from the cell body to the end of the axon, respectively. Accordingly, it is concluded that Miro specifically controls the activity of anterograde mitochondrial transport (Guo, 2005).
All Miro mutant alleles were identified by the loss of phototaxis of EGUF-induced mosaic flies; this phenotype is apparently caused by a defect in synaptic transmission, as indicated by the activity-dependent loss of Off transients in electroretinogram recordings from Miro mutant eyes (Babcock, 2003). To examine in more detail the effects of the chronic loss of mitochondria on synaptic physiology, evoked excitatory junctional potentials (EJPs) and miniature excitatory junctional potentials (mEJPs) were recorded from larval NMJs. Less than half of all Miro mutant muscles examined showed decreased resting potentials and/or a high rate of spontaneous quantal release. The remaining muscles exhibited a normal resting potential, deviating by no more than ± 5 mV from control, and a normal frequency of spontaneous release. Since the resting potential was normal for the majority of Miro muscles, partially depolarized muscles were excluded from the subsequent analysis, because their defects were likely secondary and degenerative (Guo, 2005).
Evoked EJP amplitudes of Miro mutants were normal at a low stimulation frequency of 0.2 Hz. Even during prolonged stimulation at 1 Hz for 5 min, EJP amplitudes were normal. However, during stimulation at 5 Hz, EJP amplitudes showed a steadily progressing fatigue after 90 s, and by 300 s, no more EJPs were elicited. Consistently, higher stimulation frequencies caused a progressively earlier onset of fatigue; at 10 Hz, EJPs became depressed within 10 s, and after 15-20 s, almost all stimuli failed to elicit EJPs. The activity-dependent fatigue of evoked release in Miro was not due to a failure of action potentials because electrotonically elicited EJPs showed a similar fatigue. A similar activity-dependent fatigue of evoked release was observed for transheterozygous combinations of B682 with other Miro alleles and a deletion uncovering the locus. Presynaptic expression of Miro at Miro-null mutant NMJs restored the activity-dependent fatigue of evoked release (Guo, 2005).
An activity-dependent failure of EJP amplitudes often indicates a depletion of releasable synaptic vesicles due to a defect in vesicle trafficking and/or endocytosis. However, the frequency of mEJPs at Miro mutant NMJs increased up to 5-fold during high-frequency stimulation and persisted for extended periods after stimulation. The abnormal increase in mEJP frequency was activity dependent. Prolonged stimulation at 1 Hz over 5 min did not cause an increase in the frequency of unitary quantal events at Miro mutant NMJs, while stimulation ≥5 Hz increased mEJP frequency. Hence, the activity-dependent increase in mEJP frequencies makes it unlikely that a defect in vesicle recycling causes the fatigue of evoked release. Interestingly, similar activity-dependent effects on transmitter release are observed upon acute pharmacological inhibition of mitochondrial function and are correlated with a severe loss of presynaptic Ca2+ clearance (Guo, 2005).
To test whether the activity-dependent defects in transmitter release of Miro mutants are linked to an impaired presynaptic Ca2+ homeostasis, presynaptic Ca2+ concentrations were examined at Miro NMJs. The resting [Ca2+]i was significantly elevated more than two fold in Miro mutant terminals. However, during stimulation at 80 Hz for 5 s, the peak cytosolic [Ca2+]i and the time course of [Ca2+]i decay in mutant terminals were indistinguishable from those in controls. This suggests that the chronic lack of mitochondria does not affect nerve-evoked Ca2+ levels during brief periods of stimulation. Nerve-evoked Ca2+ levels during longer periods of stimulation (10 Hz/30 s) were initially similar between control and Miro but progressively increased during stimulation to a significantly larger degree in Miro mutants, suggesting that Ca2+ buffering during extended periods of high activity is impaired (Guo, 2005).
Assuming that mitochondria in control terminals accumulate a significant amount of Ca2+ in response to nerve stimulation and subsequently slowly release it into the cytosol, one might expect that post-tetanic Ca2+ decay might be altered in the mutants, but this was not the case. In both mutants and controls, 3 s after stimulation ended, Ca2+ levels had fallen to ~8% of the plateau level achieved at the end of the stimulation train, indicating that cytosolic Ca2+ clearance is effectively no different between strains. Hence, these results suggest that cytosolic Ca2+ handling is impaired only during prolonged repetitive stimulation in Miro mutants, while fast Ca2+ clearance after stimulation is not affected. Hence, it is unlikely for two reasons that an abnormal accumulation of Ca2+ during 10 Hz stimulation causes the desynchronization of transmitter release in Miro mutant terminals: (1) stimulated Ca2+ levels only reached ~140 nM after 30 s of 10 Hz stimulation, which is in the physiological range for these terminals, and (2) Ca2+ resting levels in Miro mutant terminals before and after stimulation were indistinguishable (Guo, 2005).
The analysis of presynaptic Ca2+ dynamics in nerve terminals that chronically lack mitochondria suggests only a minor role for mitochondria as presynaptic Ca2+ sinks, which is much in contrast to the suggested dominant role of mitochondrial Ca2+ sinks at other NMJs. However, the current results could be misleading for two reasons: (1) mitochondria at larval Drosophila motor terminals may not sequester Ca2+ during stimulation; (2) other mechanisms may compensate for the chronic lack of mitochondrial Ca2+ sinks. To test the first possibility, mitochondrial Ca2+ uptake at presynaptic terminals of wild-type NMJs was examined by employing the Ca2+ indicator dihydrorhod-2 AM (rhod-2), which accumulates specifically in mitochondria (Guo, 2005).
At rest, rhod-2 produced little measurable fluorescence, presumably due to low Ca2+ levels in mitochondria since rhod-2 fluorescence below 100 nM Ca2+ is effectively invisible in vitro. Upon high-frequency nerve stimulation (80 Hz, 2 s), bright fluorescent punctae appeared that were within synaptic boutons and had the same size and distribution as live-stained mitochondria. The activity-dependent increase in the intensity of rhod-2 fluorescence was dependent on the onset, but not on the offset, of nerve stimulation; rhod-2 fluorescence faded slowly after stimulation had ceased and became invisible only after ~5 to 10 min. The slow decay of rhod-2 fluorescence is consistent with a slow release of mitochondrial Ca2+ and contrasts with the exceedingly rapid time course of decay of cytosolic Ca2+ indicators, like rhodamine-dextran or transgenically expressed G-CaMP. Hence, it is concluded that presynaptic mitochondria at Drosophila NMJs can sequester Ca2+ in response to high-frequency nerve stimulation. However, as suggested by the relatively normal Ca2+ homeostasis at Miro terminals lacking mitochondria, their significance as short-term Ca2+ sinks at these nerve terminals is either very limited or easily compensated by other mechanisms (Guo, 2005).
Author names in red indicate recommended papers.
Babcock, M.C., et al. (2003). A genetic screen for synaptic transmission mutants mapping to the right arm of chromosome 3 in Drosophila. Genetics 165: 171-183. 14504225
Benzer, S. (1967). Behavioral mutants of Drosophila isolated by countercurrent distribution. Proc. Natl. Acad. Sci. 58: 1112-1119.
David, G. (1999). Mitochondrial clearance of cytosolic Ca2+ in stimulated lizard motor nerve terminals proceeds without progressive elevation of mitochondrial matrix, J. Neurosci. 19: 7495-7506. 10460256
David G. and Barrett E. F. (2003). Mitochondrial Ca2+ uptake prevents desynchronization of quantal release and minimizes depletion during repetitive stimulation of mouse motor nerve terminals. J. Physiol. 548: 425-438. 12588898
De Vos, K. J., Sable, J., Miller. K. E. and Sheetz, M.P. (2003). Expression of phosphatidylinositol (4,5) bisphosphate-specific pleckstrin homology domains alters direction but not the level of axonal transport of mitochondria. Mol. Biol. Cell 14: 3636-3649. 12972553
Fransson, A., Ruusala A. and Aspenstrom, P. (2003). Atypical Rho GTPases have roles in mitochondrial homeostasis and apoptosis. J. Biol. Chem. 278: 6495-6502. 12482879
Frederick, R. L., et al. (2004). Yeast Miro GTPase, Gem1p, regulates mitochondrial morphology via a novel pathway. J. Cell Biol. 167: 87-98. 15479738
Gorska-Andrzejak, J., et al. (2003). Mitochondria are redistributed in Drosophila photoreceptors lacking Milton, a kinesin-associated protein. J. Comp. Neurol. 463: 372-388. 12836173
Guo, X., et al. (2005). The GTPase dMiro is required for axonal transport of mitochondria to Drosophila synapses. Neuron 47: 379-393. 16055062
Hollenbeck, P. J. (1996). The pattern and mechanisms of mitochondrial transport in axons. Front. Biosci. 1: d91-d102. 9159217
Hollenbeck, P. J. and Saxton, W. M. (2005). The axonal transport of mitochondria. J Cell Sci. 118(Pt 23): 5411-9. 16306220
Li, Z., Okamoto, K., Hayashi, Y. and Sheng, M. (2004). The importance of dentritic mitochondria in the morphogenesis and plasticity of spines and synapses. Cell 119: 873-887. 15607982
Rizzuto, R., Bernardi, P. and Pozzan, T. (2000). Mitochondria as all-round players of the calcium game. J. Physiol. 529: 37-47. 11080249
Stowers, R. S. et al. (2002). Axonal transport of mitochondria to synapses depends on Milton, a novel Drosophila protein. Neuron 36: 1063-1077. 12495622
Suzuki, S., et al. (2002). Ca2+-dependent Ca2+ clearance via mitochondrial uptake and plasmalemmal extrusion in frog motor nerve terminals. J. Neurophysiol. 87: 1816-1823. 11929903
Talbot, J. D., David, G. and Barrett, E. F. (2003). Inhibition of mitochondrial Ca2+ uptake affects phasic release from motor nerve terminals differently depending on external [Ca2+]. J. Neurophysiol. 90: 491-502. 12672777
Tanaka, Y., et al. (1998).Targeted disruption of mouse conventional kinesin heavy chain, Kif5B, results in abnormal perinuclear clustering of mitochondria. Cell 93: 1147-1158. 9657148
Tang Y., and Zucker, R. S. (1997). Mitochondrial involvement in post-tetanic potentiation of synaptic transmission. Neuron 18: 483-491. 9115741
Vale, R. D. (2003). The molecular toolbox for intracellular transport. Cell 112: 467-480. 12600311
Verstreken, P., et al. (2005). Synaptic mitochondria are critical for mobilization of reserve pool vesicles at Drosophila neuromuscular junctions. Neuron 47: 365-378. 16055061
Welte, M. A. (2004). Bidirectional transport along microtubules. Curr. Biol. 14: R525-R537. 15242636
date revised: 22 February 2005
Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.