InteractiveFly: GeneBrief

milton: Biological Overview | References

Gene name - milton

Synonyms -

Cytological map position - 27D5-27D7

Function - scaffold protein

Keywords - spermatogenesis, mitochondrial transport, oogenesis, axon transport of mitochondria in retinal neurons

Symbol - milt

FlyBase ID: FBgn0262872

Genetic map position - chr2L:7037990-7056252

Classification - HAP1 N-terminal conserved region and Milton: Kinesin associated protein

Cellular location - cytoplasmic

NCBI link: EntrezGene
milt orthologs: Biolitmine

Recent literature
Yu, Y., Lee, H.C., Chen, K.C., Suhan, J., Qiu, M., Ba, Q. and Yang, G. (2016). Inner membrane fusion mediates spatial distribution of axonal mitochondria. Sci Rep 6: 18981. PubMed ID: 26742817
In eukaryotic cells, mitochondria form a dynamic interconnected network to respond to changing needs at different subcellular locations. A fundamental yet unanswered question regarding this network is whether, and if so how, local fusion and fission of individual mitochondria affect their global distribution. To address this question, this study developed high-resolution computational image analysis techniques to examine the relations between mitochondrial fusion/fission and spatial distribution within the axon of Drosophila larval neurons. It was found that stationary and moving mitochondria undergo fusion and fission regularly but follow different spatial distribution patterns and exhibit different morphology. Disruption of inner membrane fusion by knockdown of dOpa1, Drosophila Optic Atrophy 1, not only increases the spatial density of stationary and moving mitochondria but also changes their spatial distributions and morphology differentially. Knockdown of dOpa1 also impairs axonal transport of mitochondria. But the changed spatial distributions of mitochondria results primarily from disruption of inner membrane fusion because knockdown of Milton, a mitochondrial kinesin-1 adapter, causes similar transport velocity impairment but different spatial distributions. Together, these data reveal that stationary mitochondria within the axon interconnect with moving mitochondria through fusion and fission and that local inner membrane fusion between individual mitochondria mediates their global distribution.

Yu, Y., Lee, H. C., Chen, K. C., Suhan, J., Qiu, M., Ba, Q. and Yang, G. (2016). Inner membrane fusion mediates spatial distribution of axonal mitochondria. Sci Rep 6: 18981. PubMed ID: 26742817
In eukaryotic cells, mitochondria form a dynamic interconnected network to respond to changing needs at different subcellular locations. A fundamental yet unanswered question regarding this network is whether, and if so how, local fusion and fission of individual mitochondria affect their global distribution. To address this question, high-resolution computational image analysis techniques were developed to examine the relations between mitochondrial fusion/fission and spatial distribution within the axon of Drosophila larval neurons. Stationary and moving mitochondria underwent fusion and fission regularly but followed different spatial distribution patterns and exhibited different morphology. Disruption of inner membrane fusion by knockdown of dOpa1, Drosophila Optic Atrophy 1, not only increased the spatial density of stationary and moving mitochondria but also changed their spatial distributions and morphology differentially. Knockdown of dOpa1 also impaired axonal transport of mitochondria. But the changed spatial distributions of mitochondria resulted primarily from disruption of inner membrane fusion because knockdown of Milton, a mitochondrial kinesin-1 adapter, caused similar transport velocity impairment but different spatial distributions. Together, these data reveals that stationary mitochondria within the axon interconnect with moving mitochondria through fusion and fission and that local inner membrane fusion between individual mitochondria mediates their global distribution.
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.
Li, A. Y. Z., Di, Y., Rathore, S., Chiang, A. C., Jezek, J. and Ma, H. (2023). Milton assembles large mitochondrial clusters, mitoballs, to sustain spermatogenesis. Proc Natl Acad Sci U S A 120(34): e2306073120. PubMed ID: 37579146
Mitochondria are dynamic organelles that undergo frequent remodeling to accommodate developmental needs. This study describes a striking organization of mitochondria into a large ball-like structure adjacent to the nucleus in premeiotic Drosophila melanogaster spermatocytes, which we term "mitoball". Mitoballs are transient structures that colocalize with the endoplasmic reticulum, Golgi bodies, and the fusome. Similar premeiotic mitochondrial clusters were observed in a wide range of insect species, including mosquitos and cockroaches. Through a genetic screen, Milton, an adaptor protein that links mitochondria to microtubule-based motors, mediates mitoball formation. Flies lacking a 54 amino acid region in the C terminus of Milton completely lacked mitoballs, had swollen mitochondria in their spermatocytes, and showed reduced male fertility. It is suggested that the premeiotic mitochondrial clustering is a conserved feature of insect spermatogenesis that supports sperm development.


Mitochondria are distributed within cells to match local energy demands. The microtubule-dependent transport of mitochondria depends on the ability of Milton (Milt) to act as an adaptor protein that can recruit the heavy chain of conventional kinesin-1 (kinesin heavy chain [KHC]) to mitochondria. Biochemical and genetic evidence demonstrate that kinesin recruitment and mitochondrial transport are independent of kinesin light chain (KLC); KLC antagonizes Milton's association with KHC and is absent from Milton-KHC complexes, and mitochondria are present in klc-/- photoreceptor axons. The recruitment of KHC to mitochondria is, in part, determined by the NH2 terminus-splicing variant of Milton. A direct interaction occurs between Milton and Miro, a mitochondrial Rho-like GTPase, and this interaction can influence the recruitment of Milton to mitochondria. Thus, Milton and Miro are likely to form an essential protein complex that links KHC to mitochondria for light chain-independent, anterograde transport of mitochondria (Glater, 2006).

Milton has been shown to be required for mitochondrial transport within Drosophila photoreceptors (Stowers, 2002). Mitochondria are absent from Milt photoreceptor axons, but are normally distributed and appeared to be functional in their cell bodies. Although devoid of mitochondria, their axons and synapses are otherwise surprisingly normal in their general architecture, possessing microtubules, synaptic vesicles, and active zone specializations. Thus, the transport defect is selective for mitochondria (Stowers, 2002; Gorska-Andrzejak, 2003). The mechanism of Milton's action has remained unknown, but Milton is associated with mitochondria and coimmunoprecipitates with kinesin heavy chain (KHC) in extracts of fly heads (Stowers, 2002). The mammalian homologues milton 1 and 2, which are also called O-linked N-acetylglucosamine-interacting protein 106 (OIP106) and gamma-aminobutyric acid A receptor-interacting factor-1 (GRIF-1), also colocalize with mitochondria and coimmunoprecipitate with KIF5B, which is a mammalian homologue of Drosophila KHC. Therefore, it has been suggested that Milton acts as an adaptor or regulator of the mitochondrial anterograde motor (Glater, 2006 and references therein)

This study examined the involvement of Milton in kinesin-mediated mitochondrial motility and, thus, in the essential process of distributing mitochondria within the cell. From these studies a model was derived of a protein complex that includes kinesin and adaptor proteins that link kinesin to the mitochondrion. These proteins are also likely to serve as a focal point for regulating mitochondrial motility (Glater, 2006).

In vivo, milton is required for the axonal transport of mitochondria throughout the nervous system (Stowers, 2002). Milton associates with kinesin-1 via a highly conserved domain located between residues 138 and 450. This association can recruit kinesin to mitochondria in COS7 cells and appears to activate plus end-directed transport of mitochondria, as judged by their redistribution to aggregates in the periphery of many cells transfected with both milton and KHC. These findings provide a mechanistic explanation for the absence of mitochondria from milton axons and terminals. Consistent with this model, the motors that endogenously associate with Milton, KHC in Drosophila, and KIF5 members in mammals have previously been implicated in the axonal transport of mitochondria (Glater, 2006).

The association of Milton with mitochondria appears to be mediated, in part, by its interactions with Miro, and this probably accounts for the failure of mitochondrial transport in the axons of miro mutants (Guo, 2005). This proposal is supported (a) by the ability of a truncated cytosolic form of miro to act as a dominant negative and displace milton from mitochondria, and (b) by the ability of overexpressed full-length miro to recruit to mitochondria a truncated milton (residues 1-750) that could not independently localize there. However, additional interactions for tethering milton to mitochondria are likely, as a COOH-terminal portion of milton (residues 847-1,116) also localizes to the organelle. The difficulty of purifying mitochondria from limited numbers of homozygous miro larvae prevents a direct determination of the amount of milton on mitochondria that lack miro (Glater, 2006).

The role of miro in kinesin-mediated transport does not preclude additional roles for miro. Indeed, such functions are likely because a Miro homologue, GEM1p, is found in yeast, where mitochondrial motility is chiefly actin-based, and GEM1 mutants have abnormal mitochondrial distributions. In addition, it will be of interest to determine the relationship of Milton and Miro to Syntabulin (Cai, 2005), which is another protein that has recently been proposed to link kinesin to mitochondria (Glater, 2006).

Unexpectedly, it was found that axonal transport of mitochondria did not require the light chains of the kinesin-1 motors and that light chains were, indeed, absent from the milton-kinesin complex. When expressed in COS7 and HEK293T cells, the association between Milton and KHC was inhibited by KLC. In fly homogenates, KLC was not detected in immunoprecipitates of the Milton-KHC complex. Mitochondria were abundant in the axons of klc-/- photoreceptors. Thus, this mitochondrial motor provides an exception to the conventional tetrameric structure of kinesin-1. Precedent for KHC-based transport that is KLC independent has been reported in Neurospora crassa, sea urchins, neuronal dendrites, and the transport of RNA particles (Glater, 2006).

The interaction of Milton with KHC was not only KLC independent, but was inhibited by KLC overexpression in transfected COS7 and HEK293T cells. Therefore, a pool of KHC without KLC is required for Milton to associate with KHC in vivo. Previous studies have found evidence for such a pool in bovine brain. In light of the current findings, it may be appropriate to consider the light chains as one of several cargo adaptors for kinesin-1, of which Milton is another (Glater, 2006).

Mitochondria are not static. In dividing cells they go through orchestrated movements to distribute themselves between the daughter cells. Within axons they typically alternate between stationary and moving states and can reverse their direction. They arrest in the presence of elevated Ca2+, including Ca2+ that is derived from the activation of synaptic receptors, and respond to the activation of neurotrophin receptors and various intracellular signals. It is noteworthy that, in addition to linking kinesin to the mitochondria, the Milton-Miro complex provides several possible mechanisms for the regulation of transport. These include the alternative splicing of Milton, the posttranslational modification of Milton, and the modulation of the state of miro (Glater, 2006).

The choice of NH2 terminus splicing variant can influence KHC's association with the adjacent region of Milton. In particular, KHC did not associate with Milton-C, although it contains the KHC-association domain that is common to all the isoforms. The NH2 terminus of Milton-C presumably inhibits the interaction with KHC and might, thereby, reserve a pool of mitochondria for retention in the cell body. Alternatively, the inhibition may not be constitutive in vivo, but, instead, might undergo regulation by additional factors and thereby control the recruitment of kinesin. In this context, it may be noteworthy that multiple bands of Milton are detected on immunoblots from fly heads. Most of the milton isoforms in these homogenates are in an association with KHC, as determined by immunodepletion with anti-KHC. However, there is one major band, representing nearly half of the endogenous Milton, which does not appear to be associated with KHC (Stowers, 2002). Thus, additional motors may associate with Milton, and particularly with Milton-C. Milton may also be involved in such processes as mitochondrial fission and elongation, and such a role might explain the clustering of mitochondria when Milton and Miro are overexpressed (Glater, 2006).

The alternative splicing of Milton may also represent an adaptation of the complex to the needs of particular cell types. Antiserum P1-152, which binds only to Milton-A, labels a subset of the structures in the Drosophila brain that are recognized by antibodies to the common regions (Stowers, 2002). Thus, there is tissue specificity in the choice of splicing variant. To date, ESTs for Milton-D have only been found in a testes library; therefore, Milton-D may correspond to the male-specific milton transcripts on Northern blots (Stowers, 2002) and be necessary for the elongation of mitochondria along the axoneme of sperm (Glater, 2006).

Posttranslational modifications are also likely to regulate mitochondrial motility. In particular, the COOH-terminal portions of the mammalian miltons bind to, and are substrates for, the cytosolic glycosylating enzyme O-GlcNAc transferase (OGT). Drosophila OGT has been identified by mass spectroscopy in immunoprecipitates of Milton from fly homogenates. In addition, GlcNAc-modified Milton is associated with kinesin in vivo in Drosophila, although the physiological consequences of this conserved modification are not known (Glater, 2006). Miro, however, may be of the greatest potential interest as a regulator of mitochondrial motility because it contains two predicted calcium-binding EF hands, which are flanked by two GTPase domains. Because Ca2+ stops mitochondrial movement, and thereby concentrates mitochondria near areas of high energy demand, such as active synapses, the EF hands of Miro are likely to be particularly important (Glater, 2006).

Mitochondrial motility is a feature of most, perhaps all, eukaryotic cells. In neurons, much of this motility is microtubule based, with kinesin as the plus end-directed motor. This motility, and its regulation by a variety of signals, permits the mitochondria to be distributed in accordance with local energy use. Inadequate mitochondrial function in axons or dendrites can result in decreased synapse formation, a failure to maintain synaptic transmission, or axonal degeneration. The identification of milton and miro as key components of the mechanism for mitochondrial transport by KHC should lead to a greater mechanistic understanding of the regulation of mitochondrial movement (Glater, 2006).

Milton, which has been shown to associate with Kinesin and to mediate axonal transport of mitochondria, controls the early acquisition of mitochondria by Drosophila oocytes

Mitochondria in many species enter the young oocyte en mass from interconnected germ cells to generate the large aggregate known as the Balbiani body. Organelles and germ plasm components frequently associate with this structure. Balbiani body mitochondria are thought to populate the germ line, ensuring that their genomes will be inherited preferentially. milton, a gene whose product has been shown to associate with Kinesin and to mediate axonal transport of mitochondria, is needed to form a normal Balbiani body. In addition, germ cells mutant for some milton or Kinesin heavy chain (Khc) alleles transport mitochondria to the oocyte prematurely and excessively, without disturbing Balbiani body-associated components. These observations show that the oocyte acquires the majority of its mitochondria by competitive bidirectional transport along microtubules mediated by the Milton adaptor. These experiments provide a molecular explanation for Balbiani body formation and, surprisingly, show that viable fertile offspring can be obtained from eggs in which the normal program of mitochondrial acquisition has been severely perturbed (Cox, 2006).

The Balbiani body, a large aggregate of mitochondria frequently associated with other membranous organelles and germ plasm components, is found in the newly formed oocytes of diverse species. Although it has been postulated to play a role in germ cell development and mitochondrial inheritance, no function for the Balbiani body has been demonstrated. Previously, the Drosophila Balbiani body arises when a large number of mitochondria from sister germ cells associate with the fusome, move towards its center and enter the oocyte en masse where they supplement the pre-existing mitochondria of the oocyte (Cox, 2003). Like ooctye development itself, Balbiani body formation requires the genes hts and egl, suggesting that mitochondrial movement depends on Dynein/dynactin-mediated minus-end directed transport along polarized microtubules (Cox, 2003). Studies of the mitochondrial adaptor protein Milton and its partner Kinesin heavy chain now show that plus-end directed mitochondrial transport determines when and how large a Balbiani body will form (Cox, 2006).

Mitochondrial position within cells of diverse types is frequently regulated by motor-dependent transport along microtubules. Often such positioning optimizes the ability of mitochondria to generate energy or metabolic products in appropriate subcellular locations. In Drosophila photoreceptors, neurons and in cultured cells, Milton plays a key role in positioning mitochondria by acting as a adaptor molecule between mitochondria and the Khc plus-end-directed microtubule motor. Null Khc mutations and type II milt alleles cause premature entry of an excess number of mitochondria into the oocyte. This suggests that the orchestrated movement of mitochondria within germline cysts and its sudden entry into the oocyte during follicle formation is controlled by plus-end directed transport machinery that opposes Dynein-mediated minus-end directed movement towards the oocyte. Plus-end directed activity is not needed for mitochondria to associate with the fusome; normal fusome interactions are still observed in the absence of Khc or milt function. However, the opposing action of Milt and Khc appears to be particularly effective near ring canals, especially the four oocyte ring canals, just outside of which mitochondria accumulate for a period of 1-2 days prior to follicle formation. As a new follicle prepares to bud off, an unknown modulation relieves the standoff and leads to the rapid influx of mitochondria into the oocyte where they coalesce with endogenous mitochondria to form the Balbiani body. In the absence of any movement, as in milt92 cysts, or in cysts with compromised Dhc function, a much smaller cluster of mitochondria forms in the oocyte, made up only of organelles inherited during germ cell divisions (Cox, 2006).

Complete loss of Milton did not enhance mitochondrial movement into the oocyte, as expected if its sole function was linkage to Khc. Instead, milt is needed for both minus-end directed and plus-end directed movement. Upregulation of Milt-PB relative to Milt-PA favors Dynein-based movement, but the basis for this effect remains unclear. Both Milt isoforms contain a common Kinesin binding domain, and associate with Kinesin in vivo. Evidence that Milt proteins bind Dynein directly is lacking, and the related GRIF1 protein does not bind Dynein. Thus, Milt-PB probably promotes linkage of mitochondria to Dynein indirectly, perhaps by binding and modulating Dynactin. Consistent with this view, the HAP-1 domain that differs between the two isoforms has been predicted to mediate interaction with Dynactin. Thus, changes in the relative amounts of Milt isoforms, and in their interactions with mitochondria appear to regulate the location of these organelles (Cox, 2006).

Related mechanisms may control the movement along the fusome and entry into the oocyte of other cargos besides mitochondria. Organelles such as Golgi elements, and specific mRNAs such as Bic-D, oskar and cup localize towards the center of developing 16-cell cysts, and enter the oocyte (Cox, 2003). oskar and cup RNA transiently associate with the Balbiani body in forming follicles (Cox, 2003). However, all these RNAs localize to the initial cyst cell earlier than mitochondria (Cox, 2003), and it is found that Cup continues to accumulate preferentially in the oocyte even in Dhc64C6-6/6-12 mutants that block mitochondrial transport. Consequently, even if all these components are localized based an interplay of plus-end- and minus-end-directed micotubule transport, their movement towards the oocyte is regulated differently, possibly because each is linked by cargo-specific adaptors (Cox, 2006).

Finally, these experiments provide the first test of Balbiani body function. The initial wave of mitochondria that enter the oocyte of new follicles in the Balbiani body have been proposed to have high fitness, and to represent the inheritance bottleneck of mitochondrial genomes (Cox, 2003). Oocytes from milt alleles, where this process has been strongly disrupted, still give rise to viable and fertile offspring. In part, this may be due to the observation that an independent system of mitochondrial copy number control acts to correct initial increases or deficits in oocyte mitochondrial number. Future studies will be required to determine if mitochondrial inheritance patterns are altered in milt class II mutants, and if the offspring of these alleles suffer an increased incidence of mitochondrial dysfunction over their lifespan (Cox, 2006).

Sustained elongation of sperm tail promoted by local remodeling of giant mitochondria in Drosophila

Sperm length in Drosophilidae varies from a few hundred microns to 6 cm as a result of evolutionary selection. In postcopulatory competition, longer sperm have an advantage in positioning their head closer to the egg. Sperm cell elongation can proceed in the absence of an axoneme, suggesting that a mechanism besides intraflagellar transport emerged to sustain it. This study reports that sperm elongation in Drosophila melanogaster is driven by the interdependent extension of giant mitochondria and microtubule array that is formed around the mitochondrial surface. In primary cultures of elongating spermatids, it was demonstrated that the mitochondrial integrity and local dynamics of microtubules at the tail tip region are essential for uniaxial elongation of the sperm tail. Mitochondria-microtubule linker protein Milton accumulated on mitochondria near the tail tip and is required for the sliding movement of microtubules. Disruption of Milton and its associated protein dMiro, and of potential microtubule crosslinkers Nebbish and Fascetto, caused strong elongation defects, indicating that mitochondria-microtubule association and microtubule crosslinking are required for spermatid tail elongation. It is concluded that mitochondria play unexpected roles in sperm tail elongation in Drosophila by providing a structural platform for microtubule reorganization to support the robust elongation taking place at the tip of the very long sperm tail. The identification of mitochondria as an organizer of cytoskeletal dynamics extends the understanding of mechanisms of cell morphogenesis (Noguchi, 2011).

Diversity of sperm morphology has long been a source of fascination among reproductive biologists. This diversity evolved rapidly as a result of the intense postmating reproductive selection process called 'sperm competition,' in which sperm from different males compete for a chance to fertilize an egg. This selection has driven the evolution of sperm morphologies into unique shapes that maximize the opportunity for successful fertilization. Length of Drosophilidae sperm varies by up to two orders of magnitude, reaching 6 cm (over 20 times the male body length) in the extreme case of Drosophila bifurca. There is a strong correlation between the length of the sperm and that of the seminal receptacle, the female’s sperm-storage organ, and a longer sperm tail may be advantageous for optimally positioning the sperm head for fertilization. In addition, regional diversity in the sperm and storage organ can lead to morphological incompatibility, thereby promoting reproductive isolation and speciation (Noguchi, 2011).

In the testis of D. melanogaster, right after meiosis, spherical spermatids of 10 mm in diameter, clustered in 64-cell cysts, elongate synchronously to become thin cylinders that are 0.6 mm in diameter and 1850 mm in length. After elongation, each spermatid has lengthened 185-fold and increased its surface area 11-fold without increasing its volume, suggesting that elongation requires the construction of a very long internal skeleton and the production of an extensive membrane by the cell. There are four major structures running through the longitudinal axis of an elongating spermatid: the axoneme, cytoplasmic microtubules (MTs), mitochondria, and F-actin cables. Evidence suggests that the axoneme is dispensable for sperm elongation: spermatids can grow very long in axonemeless Dsas-4 mutant flies and also in mutants in which β2-tubulin isotype is replaced with a chimeric β2β3-hybrid tubulin. Therefore, an axoneme-independent elongation mechanism is likely responsible for the extremely long sperm in Drosophilidae (Noguchi, 2011).

Insect sperm generally develop giant mitochondria that run along the length of the sperm tail. At the end of meiosis, a massive fusion of mitochondria mediated by Fuzzy onions, a testis-specific Mitofusin, leads to the two lobes of fused mitochondria being packed into a spherical structure called nebenkern. During spermatid elongation, the mitochondrial lobes unfold and elongate parallel to the axoneme to fill the entire length of the tail. It is thought that these structures extend the length of axonemeless mutant spermatids. This study reports the interdependent role of the mitochondria and cytoplasmic MTs in sperm tail elongation, which highlights a novel role for mitochondria as an organizing center of MTs and cell morphogenesis (Noguchi, 2011).

The elongation of giant mitochondria, driven by the MT bundles associated with them, serves as a cell shape template during sperm tail elongation in Drosophila melanogaster. Because of their double-membrane architecture and large size, the giant mitochondria in Drosophila spermatids possess an intrinsic stiffness. Synchronizing the 64 pairs of mitochondrial elongation events in a cyst would greatly increase the overall strength of the spermatid bundle. When mitochondrial morphogenesis was impaired in milton mutants, the spermatids became bent, suggesting that the remaining axoneme was not physically strong enough to support tail elongation. It is therefore proposed that the giant mitochondria are the main internal skeleton providing stiffness for elongating spermatid. This is the first report showing the determination of external cell morphology by mitochondria (Noguchi, 2011).

Acentrosomal mechanisms for continuous MT organization are observed in cells with long cytoplasmic extensions, such as neuronal axons, myotubes, root hair cells, and pollen tubes of plants. This study shows that in the spermatid, mitochondria organize MTs independently from the classical MTOC. The coupling of cytoskeletal array formation with an energy-producing organelle would constitute a self-sustainable system for cell morphogenesis. The special context of the giant mitochondria in Drosophila spermatids may have allowed the Milton-dependent mitochondrial transport system to acquire a novel role in cell morphogenesis (Noguchi, 2011).

This study revealed that three distinct regulations of MTs occur in the vicinity of the mitochondria: (1) new MT formation on the surface of mitochondria revealed by the MT regrowth experiment, (2) MT-mitochondria sliding mediated by MT-mitochondria crosslinker Milton/dMiro, and (3) lateral crosslinking of MTs mediated by kinesin family proteins encoded by neb, Khc73, and an antiparallel MT bundling protein encoded by feo. Through the combination of these three mechanisms, it is suggested that new MTs are formed and slide on the surface of mitochondria. Crosslinking of MTs in opposite orientations serves as a pivot point for extension or shrinkage of mitochondria when a motor protein complex crosslinking MTs and mitochondria generates a sliding force. Crosslinked parallel MTs slide together until their movement is slowed down by crosslinking to other MTs. Thus, as the number of crosslinked MTs increases, the MT array reaches a semistable tug-of-war situation, which is evident from the back-and-forth movement of EB1-GFP comets restricted within a few microns. Once a crosslinked MT network is established on the surface of mitochondria, it prevents the mitochondria from shrinking and functions as a ratchet, holding the mitochondria in an elongated shape (Noguchi, 2011).

Because spermatid elongation was sensitive to both MT-depolymerizing drug nocodazole and MT-stabilizing drug taxol and was also affected by RNAi of MT-depolymerizing kinesin Klp59D, a proper balance of MT polymerization and depolymerization is important for elongation. The local drug application experiment demonstrated that MTs at the tail tip region are essential for elongation. Furthermore, accumulation of GFP-Milton-D and sliding and rapid turnover of MTs were limited to the tail tip region, suggesting that MT-mitochondria crosslinking by a bound MT motor sustains MT sliding and growth zone. A similar relationship is seen during axon growth between dynamic MTs in the growth cone and the stable axonal MT bundle (Noguchi, 2011).

Based on the MT array-mitochondria interaction described above, a tentative model of spermatid elongation is proposed. At the tail region, the tip of elongating mitochondria provides an open surface for new MT formation. Crosslinking of antiparallel MTs stretches the mitochondria by sliding forces generated by the Milton-dMiro linker complex and exposes the membrane to create a new free surface for further MT formation and MT-mitochondria linkage. The crosslinked MTs develop into a network in a semistable tug-of-war state. Further crosslinking and slowing down of dynamics in the rear region ensure formation of highly stable MT arrays to maintain the elongated part of mitochondria. This interdependent feed-forward cycle ensures the continuous elongation of the MT array-mitochondria complex (Noguchi, 2011).

How did sperm morphogenesis change so rapidly within a relatively short period of evolution? Switching from intraflagellar transport to a mitochondria-based elongation system may have facilitated the emergence of sperm-length variation. One key feature of the mitochondria-driven mechanism is that the elongation rate is determined by active mitochondrial remodeling at the tail tip region, marked by GFP-Milton-D. Variation in sperm length is also seen in Lepidoptera, in which it correlates with the size of the sperm-storage organ in the female. The association of giant mitochondria with axoneme is a conserved characteristic of spermatids among insects, with some exceptions. Thus, the sperm-elongation mechanism described in this report might be a general system for facilitating sperm-size variation among insects, thereby enhancing sexual selection and reproductive isolation (Noguchi, 2011).

Mitofusin 2 is necessary for transport of axonal mitochondria and interacts with the Miro/Milton complex

Mitofusins (Mfn1 and Mfn2) are outer mitochondrial membrane proteins involved in regulating mitochondrial dynamics. Mutations in human Mfn2 cause Charcot-Marie-Tooth disease (CMT) type 2A, an inherited disease characterized by degeneration of long peripheral axons, but the nature of this tissue selectivity remains unknown. Evidence is presented that Mfn2 is directly involved in and required for axonal mitochondrial transport, distinct from its role in mitochondrial fusion. Live imaging of neurons cultured from Mfn2 knock-out mice or neurons expressing Mfn2 disease mutants shows that axonal mitochondria spend more time paused and undergo slower anterograde and retrograde movements, indicating an alteration in attachment to microtubule-based transport systems. Furthermore, Mfn2 disruption altered mitochondrial movement selectively, leaving transport of other organelles intact. Importantly, both Mfn1 and Mfn2 interact with mammalian Miro (Miro1/Miro2) and Milton (OIP106/GRIF1) proteins, members of the molecular complex that links mitochondria to kinesin motors. Knockdown of Miro2 in cultured neurons produced transport deficits identical to loss of Mfn2, indicating that both proteins must be present at the outer membrane to mediate axonal mitochondrial transport. In contrast, disruption of mitochondrial fusion via knockdown of the inner mitochondrial membrane protein Opa1 had no effect on mitochondrial motility, indicating that loss of fusion does not inherently alter mitochondrial transport. These experiments identify a role for mitofusins in directly regulating mitochondrial transport and offer important insight into the cell type specificity and molecular mechanisms of axonal degeneration in CMT2A and dominant optic atrophy (Misko, 2010).

Glucose regulates mitochondrial motility via Milton modification by O-GlcNAc transferase

Cells allocate substantial resources toward monitoring levels of nutrients that can be used for ATP generation by mitochondria. Among the many specialized cell types, neurons are particularly dependent on mitochondria due to their complex morphology and regional energy needs. This study reports a molecular mechanism by which nutrient availability in the form of extracellular glucose and the enzyme O-GlcNAc Transferase (OGT; termed super sex combs by FlyBase), whose activity depends on glucose availability, regulates mitochondrial motility in neurons. Activation of OGT diminishes mitochondrial motility. The mitochondrial motor-adaptor protein Milton was established as a required substrate for OGT to arrest mitochondrial motility by mapping and mutating the key O-GlcNAcylated serine residues. The GlcNAcylation state of Milton was found to be altered by extracellular glucose, and OGT was found to alter mitochondrial motility in vivo. These findings suggest that, by dynamically regulating Milton GlcNAcylation, OGT tailors mitochondrial dynamics in neurons based on nutrient availability (Pekkurnaz, 2014).

Human Miltons associate with mitochondria and induce microtubule-dependent remodeling of mitochondrial networks

Proper mitochondrial distribution is crucial for cell function. In Drosophila, mitochondrial transport is facilitated by Miro and Milton, which regulate mitochondrial attachment to microtubules via kinesin heavy chain. Mammals contain two sequence orthologs of Milton however, they have been ascribed various functions in intracellular transport. This report shows that the human Miltons target to mitochondria irrespective of whether they are linked to GFP at their C- or N-termini. Their ectopic expression induces the formation of extended mitochondrial tubules as well as large bulbous-like mitochondria with narrow tubular membrane necks that connect them to the mitochondrial mass. The mitochondrial extensions appear highly dynamic and their formation relies on the presence of microtubules. Using the photoswitchable fluorescent protein Dendra2 targeted to the mitochondrial matrix, it was found that the mitochondrial extensions and bulbous mitochondria are fused with neighboring regions of the network. Truncation analysis of huMilton1 revealed that the N-terminal region, inclusive of the coiled-coil segment could localize to microtubules, suggesting that Milton attachment to kinesin occurs independent of Miro or mitochondrial attachment. In addition, it was shown that the huMiltons have the capacity to self-interact and can also facilitate mitochondrial recruitment of a cytosolic Miro mutant. It is concluded that the human Miltons are important mediators of the mitochondrial trafficking machinery (Koutsopoulos, 2010).

Pink1 forms a multiprotein complex with Miro and Milton, linking Pink1 function to mitochondrial trafficking

Recessive mutations in Pink1 lead to a selective degeneration of dopaminergic neurons in the substantia nigra that is characteristic of Parkinson disease (see Drosophila as a Model for Human Diseases: Parkinson's disease). Pink1 is a kinase that is targeted in part to mitochondria, and loss of Pink1 function can alter mitochondrial morphology and dynamics, thus supporting a link between mitochondrial dysfunction and Parkinson disease etiology. This study reports the unbiased identification and confirmation of a mitochondrial multiprotein complex that contains Pink1, the atypical GTPase Miro, and the adaptor protein Milton. This screen also identified an interaction between Pink1 and Mitofilin. Based on previously established functions for Miro and Milton in the trafficking of mitochondria along microtubules, a role is postulated for Pink1 in mitochondrial trafficking. Using subcellular fractionation, it was shown that the overexpression of Miro and Milton, both of which are known to reside at the outer mitochondrial membrane, increases the mitochondrial Pink1 pool, suggesting a function of Pink1 at the outer membrane. Further, it was documented that Pink1 expressed without a mitochondrial targeting sequence can still be targeted to a mitochondria-enriched subcellular fraction via Miro and Milton. The latter finding is important for the interpretation of a previously reported protective effect of Pink1 expressed without a mitochondrial targeting sequence. Finally, it was found that Miro and Milton expression suppresses altered mitochondrial morphology induced by loss of Pink1 function in cell culture. These findings suggest that Pink1 functions in the trafficking of mitochondria in cells (Weihofen, 2009).

The mechanism of Ca2+ -dependent regulation of kinesin-mediated mitochondrial motility

Mitochondria are mobile organelles and cells regulate mitochondrial movement in order to meet the changing energy needs of each cellular region. Ca(2+) signaling, which halts both anterograde and retrograde mitochondrial motion, serves as one regulatory input. Anterograde mitochondrial movement is generated by kinesin-1, which interacts with the mitochondrial protein Miro through an adaptor protein, milton. Kinesin is present on all axonal mitochondria, including those that are stationary or moving retrograde. The EF-hand motifs of Miro mediate Ca(2+)-dependent arrest of mitochondria and elucidate the regulatory mechanism. Rather than dissociating kinesin-1 from mitochondria, Ca(2+)-binding permits Miro to interact directly with the motor domain of kinesin-1, preventing motor/microtubule interactions. Thus, kinesin-1 switches from an active state in which it is bound to Miro only via milton, to an inactive state in which direct binding to Miro prevents its interaction with microtubules. Disrupting Ca(2+)-dependent regulation diminishes neuronal resistance to excitotoxicity (Wang, 2009).

Roles for Drp1, a dynamin-related protein, and milton, a kinesin-associated protein, in mitochondrial segregation, unfurling and elongation during Drosophila spermatogenesis

Mitochondria undergo dramatic rearrangement during Drosophila spermatogenesis. In wild type testes, the many small mitochondria present in pre-meiotic spermatocytes later aggregate, fuse, and interwrap in post-meiotic haploid spermatids to form the spherical Nebenkern, whose two giant mitochondrial compartments later unfurl and elongate beside the growing flagellar axoneme. Drp1 encodes a dynamin-related protein whose homologs in many organisms mediate mitochondrial fission and whose Drosophila homolog is known to govern mitochondrial morphology in neurons. The milton gene encodes an adaptor protein that links mitochondria with kinesin and is required for mitochondrial transport in Drosophila neurons. To determine the roles of Drp1 and Milton in spermatogenesis, the FLP-FRT mitotic recombination system was used to generate spermatocytes homozygous for mutations in either gene in an otherwise heterozygous background. Absence of Drp1 was found to lead to abnormal clustering of mitochondria in mature primary spermatocytes and aberrant unfurling of the mitochondrial derivatives in early Drp1 spermatids undergoing axonemal elongation. In milton spermatocytes, mitochondria are distributed normally; however, after meiosis, the Nebenkern is not strongly anchored to the nucleus, and the mitochondrial derivatives do not elongate properly. This work defines specific functions for Drp1 and Milton in the anchoring, unfurling, and elongation of mitochondria during sperm formation (Aldridge, 2007).

In order to define the roles for the essential genes Drp1 and milton in mitochondrial morphogenesis during Drosophila spermatogenesis, mosaic males were generated in which some spermatocytes became homozygous for mutant alleles. Homozygosity was indicated by loss of fluorescence associated with Ubi-GFP, originally expressed in heterozygous cells from the chromosome homologous to that carrying the mutation. Haploid spermatids derived from meiotic division of homozygous mutant spermatocytes were also marked by lack of fluorescence and were used to determine the post-meiotic roles of Drp1 and milton (Aldridge, 2007).

For most genes, the genotype of a pre-meiotic spermatocyte dictates the phenotype of any haploid spermatid derived thereof, regardless of which allele the spermatid inherits. Primary spermatocytes undergo a period of extensive pre-meiotic transcription during which the mRNAs for most of the genes required for post.meiotic spermatid differentiation are transcribed. Many of these messages undergo translational repression until the times when the gene products are needed during spermiogenesis. Post-meiotic spermatids are therefore mostly dormant transcriptionally but very active translationally. Spermatids derived from spermatocytes heterozygous for a loss of function allele typically still contain wild type mRNA and/or protein and are phenotypically normal, even if the particular spermatid has only the mutant allele. This idea applies to the expression of Ubi-GFP as well; all spermatids, even those carrying only the CyO second chromosome, in the testes of Ubi-GFP/CyO heterozygous males have nuclear fluorescence. Conversely, for mutant recessive alleles of genes, spermatids will show a mutant spermiogenesis phenotype only if derived from a homozygous mutant spermatocyte. The approach for assessing the roles of Drp1 and milton in spermatocytes and spermatids is valid since homozygous mutant spermatocytes were generated simultaneously lacking Ubi-GFP and either Drp1 or Milton, and since it was possible to subsequently characterize phenotypes of the haploid spermatids derived through meiotic divisions of those cells (Aldridge, 2007).

Since both Drp1 and milton are transcribed starting in primary spermatocytes after the gonial mitotic divisions but before meiosis, the mutant clones were generated at a stage prior to the expression of any gene product; therefore, perdurance of Drp1 or Milton in mutant clones is not a significant consideration. Furthermore, the alleles of Drp1 and milton with which homozygous germline clones were made were null or strong loss of function alleles. Drp1KG03815 is a P element insertion in the first intron of Drp1 that causes lethality when homozygous and which fails to complement other lethal Drp1 alleles. The milt92 allele contains a two base pair deletion in the coding region, and the resulting frame shift leads to a truncated Milton protein of only one third the normal length. Phenotypes seen in milt92 germline clones are solely due to the milt92 mutation, since homozygous milt92 FRT40A males carrying an extra wild type copy of milton are viable and fertile with normal spermatogenesis (Aldridge, 2007).

The phenotype of mutant Drp1 primary spermatocytes is consistent with a role for Drp1 in mitochondrial fission and suggests that mitochondrial fusion and fission are normally active and counterbalanced in primary spermatocytes, as in S. cerevisiae. After gonial mitotic divisions and before meiosis, primary spermatocytes grow dramatically in size. At an early stage during this process, mitochondria temporarily aggregate before dispersing again and multiplying. While previous studies have not indicated mitochondrial fusion and fission during the primary spermatocyte stage, the abnormal retention of a tight cluster of mitochondria in mature primary spermatocytes lacking Drp1 is consistent with the possibility that (1) this cluster represents fused mitochondria that cannot divide; (2) mitochondria normally fuse in spermatocytes, perhaps on a constant basis, and especially during the early 'polar' spermatocyte stage when mitochondria aggregate; and (3) in wild type cells, active Drp1-mediated division is required to balance fusion and to separate the mitochondrial network into the individual organelles seen in mature primary spermatocytes, reported to number 150 per medial cross section (Aldridge, 2007).

While the mitochondrial fusion mediator Fzo is detectable only after meiosis, its paralog dMfn24 is likely mediating low-level mitochondrial fusion in pre-meiotic spermatocytes. In the absence of Drp1, fusion predominates and results in large mitochondrial conglomerations. The possibility cannot be ruled out that other subcellular structures may also be included in the mitochondrial clusters. The data are consistent with the abnormal clustering of mitochondria seen in the cell bodies of Drp1 mutant neurons (Aldridge, 2007).

The defect observed in Drp1 spermatocytes suggests that Drp1 is required for mitochondrial morphology at an earlier point in spermatogenesis than was originally hypothesized . Failure of putative Drp1-mediated mitochondrial fission in Drp1 mutant primary spermatocytes, and the resulting formation of an interconnected mitochondrial mass, has serious implications for the segregation of mitochondria during subsequent meiotic divisions. In wild type testes, individual mitochondria align on the meiotic spindle during each meiotic division, enabling roughly even mitochondrial distribution to daughter cells. If the mitochondrial material within a primary spermatocyte comprises an indivisible mass, then such segregation of mitochondria to secondary spermatocytes and then to spermatids should prove difficult, unless the force of cytokinetic division can trigger the breakage of the mitochondrial mass spread between daughter cells. However, the nature of meiotic cytokinesis makes such forced mitochondrial breakage unlikely, since cytoplasmic bridges between the meiotic products of a primary spermatocyte remain open throughout spermatid differentiation (Aldridge, 2007).

The configuration of mitochondria in early spermatids derived from homozygous Drp1 spermatocytes indeed suggested that the mitochondrial material began as an indivisible mass, which could not be divided properly during meiotic cytokinesis. The mitochondrial material in up to four cells at a time appeared connected, passing through the cytoplasmic connections between spermatids. In spermatid cysts whose cytoplasmic connections had been broken open by the pressure of the cover slip to give a syncytial appearance, the mitochondrial masses still appeared connected. Some mutant spermatids appeared to lack mitochondria, perhaps as a result of meiotic divisions in which the entire mitochondrial mass was segregated by chance to one of the other meiotic products of the original spermatocyte. The data suggest that Drp1 has a conserved role in mitochondrial distribution during meiosis, since the Drp1 homolog Dnm1p is required for proper mitochondrial distribution during meiosis and sporulation in the budding yeast S. cerevisiae (Aldridge, 2007).

Drp1 is required not only for mitochondrial segregation during meiosis but also perhaps for mitochondrial unfurling during axoneme elongation. In wild type cells, the two mitochondrial derivatives within a Nebenkern disentangle from each other, with the large surface area of each mitochondrial derivative immediately stretched and elongated beside the growing flagellar axoneme. It is hypothesized that Drp1-mediated mitochondrial fission would be required for the breaking of multiple topological links between the two mitochondrial derivatives within the Nebenkern during unfurling. Given that Drp1 cells are defective prior to and immediately after meiosis, the possibility cannot be definitively ruled out that the observed unfurling defects are a secondary effect of the earlier phenomena; however, secondary effects on mitochondrial unfurling are not seen in other known mutants with early mitochondrial defects. For example, an abnormally large Nebenkern initially associated with four nuclei (resulting from a meiotic cytokinesis defect) in four wheel drive mutants properly unfurls and elongates. Furthermore, in parkin and pink1 mutants, whose Nebenkerne consist of one compartment rather than two, mitochondrial unfurling still leads to a cohesive, elongating mitochondrial derivative of largely normal morphology. In Drp1 spermatids with connected Nebenkerne, one would expect normal unfurling and elongation to lead to a maximum of eight distinct linear mitochondrial derivatives, perhaps still connected. In contrast, the mitochondrial material in Drp1 spermatids spreads out, appears massively interconnected and tangled, and does not elongate. It is therefore speculated that the lack of mitochondrial fission in Drp1 spermatids may directly interfere with mitochondrial unfurling, thereby inhibiting mitochondrial elongation (Aldridge, 2007).

In neurons, both Drp1 and Milton are required for proper transport of mitochondria to synapses. However, the basis for the defects appears to be different in each case; in Drp1 homozygous neurons, a presumed failure of mitochondrial division leads indirectly to the unavailability of small transportable mitochondria, while in milton neurons, the defect appears to be more directly in the transport process. This study found that in spermatogenesis, the Drp1 and milton mutant phenotypes are distinct, confirming separate roles for these genes in mitochondrial morphogenesis (Aldridge, 2007).

The data are consistent with a role for Milton in some events of mitochondrial distribution in Drosophila spermatids. In spermatids derived from homozygous milt92 spermatocytes, Nebenkerne form properly, indicating that Milton is not required for mitochondrial aggregation. However, the Nebenkerne remain beside the nucleus only 36% of the time, compared to 89% in wild type spermatids. Milton thus contributes to proper anchoring of the Nebenkern in onion stage spermatids, though other gene products also must play a role in this attachment. In wild type cells at this stage, the Nebenkern resides directly beside the spot where the basal body is embedded in the nucleus. The axonemal microtubules emanating from the basal body are surrounded by a membraneous sheath, and the Nebenkern associates not with the axonemal microtubules directly but instead with cytoplasmic microtubules that also emanate from the basal body. Perhaps Milton, via an association with kinesin, helps connect the Nebenkern in stable fashion to cytoplasmic microtubules anchored to the nucleus (Aldridge, 2007).

Milton also plays a role in the elongation of mitochondria during axonemal growth. In wild type cells, when the two mitochondrial derivatives within a Nebenkern unfurl from each other, they very transiently appear as two round lobes (and are very rarely observed at this stage) but then are immediately distorted, pulled lengthwise into a leaf blade shape, presumably along the cytoplasmic microtubules. In spermatids derived from homozygous milt92 spermatocytes, the unfurled mitochondrial derivatives appear as spherical lobes for an extended period of time, suggesting that Milton normally mediates attachment of mitochondria to the cytoplasmic microtubules to enable shape changes. In wild type cells of a slightly later stage, the increasingly available surface area from the unfurling mitochondrial derivatives allows further elongation of the leaf blade structure. In contrast, the unfurling mitochondrial derivatives in milt92 spermatids are not immediately stretched along the cytoplasmic microtubules, remaining crumpled. This early failure of elongation occurs whether or not the mitochondrial derivatives have maintained association with the nucleus. Ultimately, some mitochondrial elongation occurs in milt92 spermatids, though mitochondrial derivatives in these cells are misshapen and usually oriented improperly with respect to the nucleus. It is concluded that Milton plays an important role in mitochondrial elongation, likely through attachment to microtubules, but that other gene products mediate some mitochondrial elongation in the absence of Milton (Aldridge, 2007).

The elongation of spermatid mitochondria may involve either (1) mitochondrial anchoring at the proximal (minus) end of cytoplasmic microtubules and subsequent sliding of mitochondrial membranes toward the distal (plus) end, or (2) progressive immobilization of mitochondrial membranes along growing cytoplasmic microtubules, analogous to the closing of a zipper. Milton (and perhaps kinesin) may act as they do in neurons, mediating mitochondrial movement toward the microtubule plus ends, or may serve simply to anchor mitochondria in static fashion during elongation. The decreased association of Nebenkerne with nuclei in milt92 onion stage spermatids also suggests an anchoring role for Milton at the microtubule minus end. The dynein motor protein has recently been shown to act not only as a progressive motor but also as a static anchor for cargo. Consistent with the bidirectional transport model, the non-kinesin-associated Milton isoform and/or the testis-specific isoform may enable anchoring or minus-end directed movement of mitochondria toward the nucleus, while other isoforms may mediate plus.end directed mitochondrial elongation (Aldridge, 2007).

The technique for generating germline clones of Drp1 and milton allowed assessment of mutant phenotypes through the mid-elongation stages of spermatogenesis, after which point the condensed nuclei and the bundled nature of the elongating sperm (sixty four cells per cyst) made it impossible to identify individual mutant cells within a cyst. Transheterozygous male flies did not have homozygous clones that encompassed entire cysts. Most structural defects during spermatogenesis cause sterility through failure of individualization, which is the final investment of each sperm with its own plasma membrane and concomitant disposal of waste materials from each cell. Given the severity of the Drp1 and milton phenotypes, it is likely that individualization of mutant sperm similarly fails in these mutants. Observations that homozygous milton germline clones in a heterozygous dominant male sterile background do not confer fertility, while clones of the background chromosome do, indeed suggest that milton sperm either fail to individualize or are non-motile due to the mitochondrial defects (Aldridge, 2007).

In summary, this study has defined roles for Drp1 and Milton in the specialized mitochondrial morphogenesis that takes place during spermatogenesis in Drosophila. Drp1-mediated mitochondrial division enables proper mitochondrial distribution during male meiosis as well as post-meiotic unfurling of mitochondrial derivatives (either directly or indirectly). Milton helps anchor the Nebenkern to the nucleus and subsequently mediates elongation of mitochondrial derivatives in developing spermatids. This work demonstrates that similar mechanisms for mitochondrial morphogenesis have been adapted for highly specialized use in different tissues within the organism (Aldridge, 2007).

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. It is proposed that Milton is a mitochondria-associated protein required for kinesin-mediated transport of mitochondria to nerve terminals (Stowers, 2002).

Neurons require the active transport of proteins and organelles over large distances to their terminals. Whereas synaptic vesicles are exclusively transported to the terminal, mitochondria present an additional challenge to the cell insofar as some must be sent down the axon while others are retained in the cell body. The milton gene is essential for the proper localization of mitochondria to the axon and terminal; in neurons homozygous for milton, mitochondria are restricted to the cell soma, whereas synaptic vesicles continue to enter axons and accumulate at synapses that retain normal anatomical specializations and appropriate contacts. This phenotype might arise from several mechanisms: a failure of the mitochondria to couple to the motor for transport, a failure in the regulation of the motor protein, or a failure of mitochondria to be retained in the terminal once they arrive there. The current findings are most consistent with the hypothesis that Milton acts as an adaptor protein or as part of an adaptor complex that links the appropriate kinesin motor to mitochondria. (1) Milton copurifies with mitochondria but is not observed on the plasma membrane or synaptic vesicles. (2) When expressed in HEK293T cells, Milton localizes selectively to mitochondria. (3) Milton colocalizes in vivo with a mitochondrial marker in Drosophila nerves. (4) Milton and kinesin are associated with one another in immunoprecipitation experiments and colocalize in HEK293T cells. (5) Mitochondria were not observed in homozygous mutant axons; if Milton were necessary to retain mitochondria in the terminal rather than to transport them, numerous mitochondria should be seen in transit in the axons. (6) Overexpression of Milton in HEK293T cells disrupted the normal distribution of mitochondria within the cell. The milton electroretinogram (ERG) phenotype appears to result from a failure of photoreceptor neurons to transport mitochondria to the nerve terminal and not to a more general mitochondrial defect. Thus, morphologically normal mitochondria in appropriate numbers are observed in the rhabdomere region of these same photoreceptors and are likely to function properly because these cells sustain normal phototransduction (Stowers, 2002).

The nature of the interactions between Milton and both mitochondria and Kinesin is not known. Milton lacks a predicted transmembrane domain and therefore may bind to a membrane protein present on the mitochondria of both Drosophila neurons and HEK293T cells. Although Milton and Kinesin coprecipitate, attempts to demonstrate a direct interaction between them with proteins expressed in vitro and by yeast two-hybrid tests were not successful. It thus appears likely that at least one additional linker protein may be required to associate mitochondria with this motor. A precedent for a protein complex serving as an adaptor exists in mLin2, mLin7, and mLin10, which form the probable adaptor for vesicles delivering NMDA receptors to dendrites. Alternatively, it remains possible that Milton is not the adaptor per se, but rather a protein that regulates independently bound Kinesin to the mitochondrion. Some kinesins, for example, may be bound via a combination of protein factors and direct interactions with membrane lipids. Furthermore, anti-KHC antibodies preferentially precipitate only a subset of Milton isoforms, suggesting that Milton may also function independently of KHC. Such roles could include tethering mitochondria at discrete energy-requiring locations within the nerve terminal or linking mitochondria to other kinesin-like motors. An indirect role for Milton in synaptic plasticity could arise from the involvement of mitochondria in regulating calcium concentrations in the nerve terminal. Nonneuronal functions concerning mitochondrial movement or localization are also suggested by the early embryonic expression of Milton before neurons arise, which may reflect mitochondrial motility during cellularization (Stowers, 2002).

Overexpression of a component of a dynein adaptor complex, dynamitin, exerts a dominant-negative effect on transport by dynein, disrupting retrograde axonal transport and causing cargoes to accumulate at the plus end of microtubules. In this context, it is interesting to note that a redistribution of the mitochondria was observed in HEK293T cells when Milton was overexpressed; the mitochondria became tightly clustered in the vicinity of the Golgi apparatus, a structure associated with the minus ends of microtubules. The redistribution may result from a comparable inhibition of kinesin, the plus end-directed motor, causing the mitochondria to accumulate at the minus ends (Stowers, 2002).

No discernible sequence motif or structural characteristic unites the adaptor proteins that have so far been identified. In addition to mLin2, mLin7, and mLin10, these include Sunday Driver, which functions on G58K-marked Golgi and post-Golgi vesicles and β-1 adaptin, which is associated with post-Golgi vesicles bearing the mannose-6-phosphate receptor. The observations that mitochondria move along axons at speeds similar to those of known kinesins and that mitochondria accumulate in the axons of Drosophila with mutant kinesin heavy chain together predict the existence of a mitochondria-specific kinesin-adaptor in neurons. However, no such mitochondrial protein has been identified (Stowers, 2002).

It is interesting that there are two Milton homologs in the human genome but none in the reported genomes of nematodes, plants, or yeast. In these species, no genes encode proteins with >25% identity to the coiled-coil region of Milton or any significant homology with the more highly conserved C-terminal homology region. These organisms lack the lengthy axons and complex cellular structures of arthropods and vertebrates and may not therefore need the active transport of mitochondria. The yeast tropomyosin 1 gene (tpm1), shows 25% identity over 148 amino acids in the coiled-coil domain of Milton and exhibits a mitochondria distribution phenotype at cell division. Possibly, therefore, Milton evolved from a Tpm1-like protein to take on a role in axonal transport of mitochondria (Stowers, 2002).

The initial observation leading to the isolation of milton, that the behavior and ERG of flies with homozygous milton photoreceptors indicated a defect in transmission by photoreceptors to downstream neurons, must be a consequence of the pivotal finding that these milton photoreceptors lack mitochondria at their terminals. Precisely how the absence of mitochondria impairs signaling, however, is less certain. The otherwise surprisingly normal ultrastructure of milt186/milt186 terminals implies that the physiological defect is not a symptom of degeneration at the terminal and that some metabolic needs of the terminal are apparently met by other energy sources such as anaerobic metabolism and the diffusion of ATP from the soma. Although the affected terminals may possibly release some neurotransmitter, the extent or synchronization of any release must be sufficiently altered to result in poor phototaxis and the absence of on- and off-transients in the ERG (Stowers, 2002).

Why, then, are Drosophila photoreceptor nerve terminals dysfunctional in the absence of mitochondria? The absence of mitochondria and consequent decrease in ATP supply could adversely affect any number of ATP-dependent processes in the terminal or axon including: the vesicular proton pump that provides the energy to load vesicles with neurotransmitter; the Na+/Ca2+ exchanger that extrudes Ca2+ from the nerve terminal; the Na+/K+-ATPase; or NSF, which facilitates the vesicle cycle by dissociating SNARE proteins. Because the population of vesicles in the terminals is not severely depleted, energy-dependent steps of endocytosis, including dynamin-dependent fission or clathrin-uncoating, seem not to be greatly compromised (Stowers, 2002).

Ca2+ homeostasis in the terminal may be particularly compromised by the absence of mitochondria. Not only will the extrusion of Ca2+ across the plasmalemma be diminished if the Na+ gradient runs down, but additionally, given that mitochondria take up Ca2+ after a Ca2+ spike, the absence of mitochondria would increase residual cytosolic Ca2+. This accumulation of Ca2+ could gradually increase neurotransmitter release even in the absence of stimulation. It is therefore possible that the absence of on- and off-transients in the ERG results not from an inability to release neurotransmitter, but rather from a constant release that is independent of light (Stowers, 2002).

The findings, that Milton is associated with Kinesin heavy chain and is required for axonal transport of mitochondria, now make it possible to address a number of questions. How does Milton attach to mitochondria and to Kinesin, and does Milton's attachment invariably destine a mitochondrion to be transported down the axon? How is the appropriate number of mitochondria in a nerve terminal established? Does nerve terminal activity alter the number of mitochondria present via Milton? It is hoped that the identification of Milton as an essential element of mitochondrial localization will make these questions accessible to biochemical study (Stowers, 2002).


Search PubMed for articles about Drosophila Milton

Aldridge, A. C., et al. (2007). Roles for Drp1, a dynamin-related protein, and milton, a kinesin-associated protein, in mitochondrial segregation, unfurling and elongation during Drosophila spermatogenesis. Fly (Austin) 1(1): 38-46. PubMed ID: 18690063

Cai, Q., Gerwin, C. and Sheng. Z. H. (2005). Syntabulin-mediated anterograde transport of mitochondria along neuronal processes. J. Cell Biol. 170: 959-969. 16157705

Cox, R. T. and Spradling, A. C. (2003). A Balbiani body and the fusome mediate mitochondrial inheritance during Drosophila oogenesis. Development 130: 1579-1590. PubMed ID: 12620983

Cox, R. T. and Spradling, A. C. (2006). Milton controls the early acquisition of mitochondria by Drosophila oocytes. Development 133(17): 3371-7. PubMed ID: 16887820

Glater, E. E., Megeath, L. J., Stowers, R. S. and Schwarz, T. L. (2006). Axonal transport of mitochondria requires Milton to recruit kinesin heavy chain and is light chain independent. J. Cell Biol. 173(4): 545-57. 16717129

Gorska-Andrzejak, J., Stowers, R. S., Borycz, J., Kostyleva, R., Schwarz, T. L. and Meinertzhagen, I. A. (2003). Mitochondria are redistributed in Drosophila photoreceptors lacking milton, a kinesin-associated protein. J. Comp. Neurol. 463(4): 372-88. 12836173

Guo, X., et al. (2005). The GTPase dMiro is required for axonal transport of mitochondria to Drosophila synapses. Neuron. 47: 379-393. 16055062

Koutsopoulos, O. S., et al. (2010). Human Miltons associate with mitochondria and induce microtubule-dependent remodeling of mitochondrial networks. Biochim. Biophys. Acta 1803(5): 564-74. PubMed ID: 20230862

Misko, A., et al. (2010). Mitofusin 2 is necessary for transport of axonal mitochondria and interacts with the Miro/Milton complex. J. Neurosci. 30(12): 4232-40. PubMed ID: 20335458

Noguchi, T., Koizumi, M. and Hayashi, S. (2011). Sustained elongation of sperm tail promoted by local remodeling of giant mitochondria in Drosophila. Curr. Biol. 21(10): 805-14. PubMed ID: <21549602

Pekkurnaz, G., Trinidad, J. C., Wang, X., Kong, D. and Schwarz, T. L. (2014). Glucose regulates mitochondrial motility via Milton modification by O-GlcNAc transferase. Cell 158: 54-68. PubMed ID: 24995978

Stowers, R. S., et al. (2002). Axonal transport of mitochondria to synapses depends on milton, a novel Drosophila protein. Neuron 36:1063-1077. 12495622

Wang, X, and Schwarz, T. L. (2009). The mechanism of Ca2+ -dependent regulation of kinesin-mediated mitochondrial motility. Cell 136(1): 163-74. PubMed ID: 19135897

Weihofen, A., et al. (2009). Pink1 forms a multiprotein complex with Miro and Milton, linking Pink1 function to mitochondrial trafficking. Biochemistry 48(9): 2045-52. PubMed ID: 19152501

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date revised: 15 December 2023

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