InteractiveFly: GeneBrief

atlastin: Biological Overview | References


Gene name - atlastin

Synonyms -

Cytological map position - 96A13-96A13

Function - transmembrane protein

Keywords - ER biogenesis and maintenance, homotypic membrane fusion, vesicle fusion

Symbol - atl

FlyBase ID: FBgn0039213

Genetic map position - 3R:20,451,397..20,460,436 [+

Classification - Guanylate-binding protein, N-terminal domain

Cellular location - transmembrane



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Liu, T. Y., Bian, X., Romano, F. B., Shemesh, T., Rapoport, T. A. and Hu, J. (2015). Cis and trans interactions between Atlastin molecules during membrane fusion. Proc Natl Acad Sci U S A 112: E1851-1860. PubMed ID: 25825753
Summary:
Atlastin (ATL), a membrane-anchored GTPase that mediates homotypic fusion of endoplasmic reticulum (ER) membranes, is required for formation of the tubular network of the peripheral ER. How exactly ATL mediates membrane fusion is only poorly understood. This study shows that fusion is preceded by the transient tethering of ATL-containing vesicles caused by the dimerization of ATL molecules in opposing membranes. Tethering requires GTP hydrolysis, not just GTP binding, because the two ATL molecules are pulled together most strongly in the transition state of GTP hydrolysis. Most tethering events are futile, so that multiple rounds of GTP hydrolysis are required for successful fusion. Supported lipid bilayer experiments show that ATL molecules sitting on the same (cis) membrane can also undergo nucleotide-dependent dimerization. These results suggest that GTP hydrolysis is required to dissociate cis dimers, generating a pool of ATL monomers that can dimerize with molecules on a different (trans) membrane. In addition, tethering and fusion require the cooperation of multiple ATL molecules in each membrane. A comprehensive model is proposed for ATL-mediated fusion that takes into account futile tethering and competition between cis and trans interactions.

Summerville, J., Faust, J., Fan, E., Pendin, D., Daga, A., Formella, J., Stern, M. and McNew, J. A. (2016). The effects of ER morphology on synaptic structure and function in Drosophila melanogaster. J Cell Sci [Epub ahead of print]. PubMed ID: 26906425
Summary:
Hereditary Spastic Paraplegia (HSP) is a set of genetic diseases caused by mutations in one of 72 genes that results in age-dependent corticospinal axon degeneration accompanied by spasticity and paralysis. Two genes implicated in HSPs encode proteins that regulate ER morphology. Atlastin (SPG3A) encodes an ER membrane fusion GTPase and Reticulon 2 (SPG12) helps shape ER tube formation. This study used a new fluorescent ER marker to show that the ER within wildtype Drosophila motor nerve terminals forms a network of tubules that is fragmented and made diffuse by atl loss. atl or Rtnl1 loss decreases evoked transmitter release and increases arborization. Similarly to other HSP genes, atl inhibits bone morphogenetic protein (BMP) signaling, and loss of atl causes age-dependent locomotor deficits in adults. These results demonstrate a critical role for ER in neuronal function and identify mechanistic links between ER morphology, neuronal function, BMP signaling, and adult behavior.
Rao, K., Stone, M. C., Weiner, A. T., Gheres, K. W., Zhou, C., Deitcher, D. L., Levitan, E. S. and Rolls, M. M. (2016). Spastin, atlastin and ER relocalization are involved in axon, but not dendrite, regeneration. Mol Biol Cell 27(21):3245-3256. PubMed ID: 27605706
Summary:
Mutations in over 50 genes including spastin and atlastin lead to Hereditary Spastic Paraplegia (HSP). It was previously demonstrated that reduction of spastin leads to a deficit in axon regeneration in a Drosophila model. Axon regeneration was similarly impaired in neurons when HSP proteins atlastin, seipin and spichthyin were reduced. Impaired regeneration was dependent on genetic background, and was observed when partial reduction of HSP proteins was combined with expression of dominant-negative microtubule regulators, suggesting HSP proteins work with microtubules to promote regeneration. Microtubule rearrangements triggered by axon injury were, however, normal in all genotypes. Other markers were examined to identify additional changes associated with regeneration. While mitochondria, endosomes and ribosomes did not exhibit dramatic repatterning during regeneration, the endoplasmic reticulum (ER) was frequently concentrated near the tip of the growing axon. In atlastin RNAi and spastin mutant animals, ER accumulation near single growing axon tips was impaired. ER tip concentration was only observed during axon regeneration, and not during dendrite regeneration. In addition, dendrite regeneration was unaffected by reduction of spastin or atlastin. It is proposed that the HSP proteins Spastin and Atlastin promote axon regeneration by coordinating concentration of the ER and microtubules at the growing axon tip.

BIOLOGICAL OVERVIEW

Establishment and maintenance of proper architecture is essential for endoplasmic reticulum (ER) function. Homotypic membrane fusion is required for ER biogenesis and maintenance, and has been shown to depend on GTP hydrolysis. This study demonstrates that Drosophila Atlastin - the fly homologue of the mammalian GTPase atlastin 1 involved in hereditary spastic paraplegia - localizes on ER membranes and that its loss causes ER fragmentation. Drosophila Atlastin embedded in distinct membranes has the ability to form trans-oligomeric complexes and its overexpression induces enlargement of ER profiles, consistent with excessive fusion of ER membranes. In vitro experiments confirm that Atlastin autonomously drives membrane fusion in a GTP-dependent fashion. In contrast, GTPase-deficient Atlastin is inactive, unable to form trans-oligomeric complexes owing to failure to self-associate, and incapable of promoting fusion in vitro. These results demonstrate that Atlastin mediates membrane tethering and fusion and strongly suggest that it is the GTPase activity that is required for ER homotypic fusion (Orso, 2009).

The ER is composed of a network of interconnected tubules that pervades the cytoplasm of eukaryotic cells. Homotypic membrane fusion is essential for ER establishment and maintenance. This activity requires GTP hydrolysis and non-cytosolic factors, suggesting the involvement of an as yet unidentified GTP-dependent fusion machinery associated with the ER membrane. Human atlastin GTPase 1 (ATL1), the mutation of which causes a form of hereditary spastic paraplegia, belongs to the dynamin superfamily of GTPases (Zhao, 2001) and has been implicated in ER–Golgi vesicle trafficking and Golgi morphogenesis (Sanderson, 2006; Zhu, 2003). Two other atlastins (Zhu, 2003) have been identified in mammals, but their function remains unexplained. Drosophila contains a single highly conserved atlastin orthologue (Atlastin). The atlastin-null mutant flies (atl1) are abnormally small and have motor dysfunction (Lee, 2008), however the cellular role of Atlastin has not been explored. This study reports that Atlastin localizes to ER membranes and that the loss of Atlastin function causes fragmentation of the ER network. Atlastin localized on adjacent membranes can form trans-oligomeric complexes, and its overexpression results in the formation of expanded ER elements, consistent with excessive fusion of membranes. Notably, Atlastin is autonomously capable of promoting in vitro fusion of liposomes. In contrast, the expression of GTPase-deficient Atlastin has no phenotypic consequences, cannot form trans-oligomeric complexes, and fails to drive membrane fusion in vitro. These results indicate that Atlastin is the crucial GTPase mediating homotypic fusion of ER membranes (Orso, 2009).

Drosophila and human Atlastins show remarkable homology and conservation of domain organization, probably resulting in analogous membrane topology (Zhu, 2003: Rismanchi, 2008). Immunohistochemistry experiments showed that Drosophila Atlastin is ubiquitously expressed, and its expression levels are high during embryonic development. In embryos, Atlastin immunoreactivity consistently overlapped with the ER-reporter green fluorescent protein (GFP)-KDEL and the ER-localized proteins BiP and Rtnl1- GFP. Simultaneous visualization of endogenous Atlastin and the Golgi apparatus using the Golgi reporter GalT-GFP or antibodies to the Golgi marker p120 demonstrate that Atlastin does not share the punctate distribution that is typical of Golgi in Drosophila. Moreover, quantitative immunogold electron microscopy (EM) analysis in Drosophila S2 cells showed that approximately 60% of gold particles were found on ER membranes. Together, these results establish that Atlastin is a membrane protein residing in the ER (Orso, 2009).

Secretory pathway traffic has been reported to be normal under knockdown conditions for all human atlastins (Rismanchi, 2008). Similarly, loss of Drosophila Atlastin did not alter mCD8-GFP membrane transport, had no effects on Golgi morphology and distribution, and did not perturb ER exit sites, indicating that Atlastin is unlikely to participate in secretory traffic. To gain insight into the function of Atlastin, the consequences of downregulating its expression in Drosophila by in vivo RNA interference (RNAi) was examined. UAS-atl-RNAi transgenic flies, the expression of which can be controlled using the Gal4-UAS system, have been generated. Several experiments demonstrated that in vivo atlastin knockdown was specific and effective. Loss of Atlastin in muscle and neurons resulted in modest changes in GFP-KDEL fluorescence; however, ultrastructural analysis of ER morphology showed considerable alterations. Control neurons had long tubular ER profiles (average length 876 nm), whereas neurons lacking Atlastin showed severely undersized ER profiles (average length 308 nm). Furthermore, the ER profile size distribution in atlastin RNAi neurons showed two classes of short ER profiles (0-200 and 200-400 nm) that were virtually absent from the cytoplasm of wild-type neurons. EM analysis showed analogous shortened ER profiles in atl1 mutant neurons, and in tubulin-Gal4/+;UAS-atl-RNAi/+ muscles. In addition, the expression of Atlastin in the null mutant background fully rescued ER fragmentation. These results indicate that the ER is fragmented in response to loss of Atlastin (Orso, 2009).

The functional consequences of these morphological changes in ER structure were examined by fluorescence loss in photobleaching (FLIP). The loss of a fluorescent marker from a region of a cell after repeated photobleaching of a different region indicates continuity between the two regions. FLIP experiments targeting GFP-KDEL were used to assess whether fragmentation in atlastin RNAi tissue resulted in discontinuity of normally interconnected ER elements. Unlike in control muscle where the loss of GFP-KDEL fluorescence was homogeneous, repetitive photobleaching of GFP-KDEL in tubulin-Gal4/+;UAS-atl-RNAi/+ muscle produced regions of unbleached fluorescence, indicating that in these areas the ER network lacked its typical continuity. Fluorescence loss was still detectable in other areas suggesting that fragmentation was partial, probably because it is the result of deterioration of ER maintenance. These data reinforce EM observations demonstrating that the removal of Atlastin results in ER fragmentation (Orso, 2009).

Because a reduction in Atlastin levels produced ER fragmentation, it was predicted that the overexpression of Atlastin may lead to excessive membrane fusion if Atlastin were involved in this process. Immunofluorescence analysis of larva brains overexpressing Atlastin-Myc with the motoneuron driver D42-Gal4 showed that the ER marker BiP accumulated in cytoplasmic structures that were absent in controls. These structures contained Atlastin and the Golgi marker p120, suggesting that Atlastin-Myc localizes properly but alters ER morphology and disrupts the Golgi apparatus. EM analysis of D42-Gal4/+;UAS-atlastin-myc/+ brains showed that Atlastin overexpression disrupts the ER network. Normal tubular ER profiles were absent in Atlastin-overexpressing motoneurons where ER membranes formed expanded cisternae. This expansion of ER elements is consistent with an increase in membrane fusion and suggests that Atlastin itself could directly mediate bilayer merger. In agreement with immunofluorescence data, normal Golgi complexes were essentially absent in neurons overexpressing Atlastin. The absence of normal Golgi and the redistribution of Golgi proteins to the ER suggest a block in secretory traffic, indicating that hyperfusion of ER membranes impairs this process (Orso, 2009).

Because human atlastin proteins self-assemble into oligomeric complexes ( Zhu, 2003; Rismanchi, 2008), attempts were made to establish whether Atlastin was also capable of homo-oligomerization. HeLa cells were simultaneously transfected with Atlastin-Myc and Atlastin-haemagglutinin (HA) constructs, and co-immunoprecipitation experiments demonstrated self-association of Atlastin molecules. This finding, together with the observations that human atlastins are transmembrane proteins and that Atlastin localizes to ER membranes and its downregulation triggers ER fragmentation, raised the possibility that Atlastin could be directly involved in tethering adjacent ER membranes, thereby permitting homotypic fusion to occur. To test this hypothesis, membrane vesicles were prepared from HeLa cells separately transfected with Atlastin-Myc or Atlastin-HA. Transfected cells were homogenized in the absence of detergent and fragmented membranes were vesiculated by sonication, mixed and immunoprecipitated. When anti-Myc antibodies were used to precipitate Atlastin-Myc-containing vesicles, Atlastin-HA was recovered in the pellet. Fractionation of cleared cell homogenates showed that Atlastin and the ER resident integral membrane protein calnexin partitioned exclusively to the membrane fraction, whereas the ER-luminal protein PDI remained in the soluble fraction. This demonstrates that under these lysis conditions, Atlastin associated with membranes and Atlastin binding occurred exclusively between distinct vesicles. Thus, Atlastin molecules inserted in adjacent ER membranes can form trans-complexes. These results establish that homophilic interactions between Atlastin molecules provide a tethering step between opposing ER membranes, leading to bilayer merger potentially mediated directly by Atlastin (Orso, 2009).

A direct role for Atlastin in membrane fusion was tested in vitro. Recombinant glutathione S-transferase (GST)-Atlastin was reconstituted into two populations of preformed liposomes and fusion was measured by lipid-mixing. Acceptor proteoliposomes contained unlabelled lipids and donor proteoliposomes contained these lipids as well as 7-nitrobenzoxadiazole (NBD) and rhodamine head-group-labelled lipids. NBD and rhodamine form a fluorescence resonance energy transfer (FRET) pair, and fusion is measured as an increase in NBD fluorescence over time as lipid mixing between donor and acceptor proteoliposomes dilutes the fluorescence probes in the newly merged membrane. When equimolar amounts of fluorescently labelled and unlabelled Atlastin proteoliposomes are mixed, magnesium and GTP addition drives robust fusion that is completely dependent on GTPase activity and independent of lipid composition. No fusion is seen using GDP, GMP, ATP or when magnesium is replaced with calcium, and fusion driven by Atlastin results in complete mixing of both inner and outer phospholipid monolayers. Atlastin-mediated fusion is concentration-dependent and correlates with an increase in size of the proteoliposome population as measured by dynamic light scattering. Furthermore, analysis of negative stained Atlastin proteoliposomes by EM showed a homogenous population of liposomes before and after fusion. These data demonstrate that Atlastin alone is sufficient to drive membrane fusion in vitro (Orso, 2009).

It was postulated that Atlastin-mediated tethering and fusion of ER membranes should rely on the GTPase activity of Atlastin. To test this hypothesis, transgenic flies were generated for the expression of Atlastin carrying a Lys51Ala substitution (UAS-atlastin-(K51A)-myc) in the P-loop of the GTP-binding domain. Biochemical analysis of recombinant wild-type and mutant (Lys51Ala) Atlastin indicates that replacement of Lys 51 significantly reduces GTPase activity (Praefcke, 2004). Consistent with this notion, GMR-Gal4 driven eye-specific expression of UAS-atlastin-(K51A)-myc had no phenotypic effects, whereas the expression of UAS-atlastin-myc gave rise to a small eye. Moreover, in contrast to overexpression of wild-type Atlastin, overexpression of Atlastin(K51A) with all drivers allowed survival of the flies, indicating that replacement of Lys 51 with Ala results in the inactivation of the protein. Notably, immunofluorescence and ultrastructural analyses of motor neurons expressing Atlastin(K51A)-Myc under the control of D42-Gal4 showed that the mutant properly localized to the ER, and ER and Golgi morphology was unaltered. Moreover, Atlastin(K51A) was unable to rescue ER fragmentation in atl1 neurons. These observations demonstrate that formation of aberrant ER depends crucially on the GTPase activity of Atlastin (Orso, 2009).

To understand how the loss of GTPase activity might impair Atlastin function, the ability of the GTPase-deficient Atlastin(K51A)-HA and Atlastin(K51A)- Myc to homo-oligomerize was assayed in co-transfected HeLa cells, as well as in the membrane-vesicle immunoprecipitation assay. In both assays, Atlastin(K51A) was unable to self-associate, indicating that this inability prevented the formation of trans-oligomeric complexes between Atlastin(K51A)-HA- and Atlastin(K51A)-Myc-containing vesicles. Thus, GTPase activity is critical for self-association and GTPase-deficient Atlastin(K51A) lacks the competence to mediate membrane tethering (Orso, 2009).

Given that Atlastin(K51A) was unable to oligomerize, its ability to drive fusion was tested. Donor and acceptor proteoliposomes containing equal amounts of Atlastin(K51A) or wild-type Atlastin were prepared and their ability to fuse was analysed. When wild-type Atlastin was incorporated in both membranes, fusion proceeded normally; however, the inclusion of Atlastin(K51A) in the reaction failed to support fusion. This result demonstrates that a functional GTPase domain is required on both membranes for fusion to occur (Orso, 2009).

Establishment of the ER network occurs by a basic homotypic fusion reaction that requires GTP hydrolysis and membrane-associated factors. The formation of a tubular network then ensues that relies on the action of cytosolic protein components. Although reticulons are the major players in tubularization of the ER, no obvious candidates responsible for mediating homotypic fusion of ER membranes have been identified (Orso, 2009).

In this study, combined in vivo and in vitro analyses provide strong evidence that Atlastin is the vital GTPase required for homotypic fusion of ER membranes. Atlastin resides in the ER -- a localization consistent with a role in mediating homotypic interactions between ER membranes. In response to loss of atlastin, the ER network becomes fragmented but no obvious transport impairment was observed, supporting a function in the maintenance of ER integrity rather than in secretory traffic. Nevertheless, subtle transport defects after fragmentation cannot be completely ruled out. Reduced membrane traffic in Drosophila results in cell growth defects, indicating that minor transport impairment secondary to ER disorganization may explain the small size of Atlastin mutant cells and individuals. Atlastin is capable of homo-oligomerization and self-association can occur within the same membrane as well as between opposing membranes. This property leads to the formation of trans-complexes that tether adjacent ER membranes. In vivo overexpression of Atlastin results in the expansion of ER elements, consistent with excessive membrane fusion. In agreement with this interpretation of in vivo overexpression results, recombinant Atlastin potently drives membrane fusion in vitro in a GTP-dependent manner. Atlastin requires GTPase activity to exert its function because GTPase-deficient Atlastin(K51A) is functionally inactive in vivo, fails to tether ER membranes owing to its inability to homo-oligomerize, and does not promote membrane fusion in vitro. It is likely that the inability of Atlastin(K51A) to fuse is directly related to its inability to self-associate (Orso, 2009).

Approximately 50% of the known dominant forms of pure hereditary spastic paraplegia have been linked to ER dysfunction. A properly functioning ER is absolutely required for all cells, given that it is the initial entry point into the secretory pathway for most secreted proteins and membrane proteins localized to the plasma membrane. Disturbance in the function or loss of integrity of the ER could result in a failure of protein folding, glycosylation or transport, leading to ER stress that may ultimately contribute to the pathogenesis of neurodegenerative disorders. Independently of their mechanism, pathological mutations in ATL1 are likely to perturb membrane fusion, with loss of ER integrity suggesting that progressive axonal degeneration in ATL1-linked hereditary spastic paraplegia patients may be the consequence of ER stress engendered by this perturbation (Orso, 2009).

These studies have uncovered a requirement for Drosophila Atlastin in the homotypic fusion of ER membranes, suggesting that Atlastin represents the GTPase activity thought to be required for this process. Although further studies will be necessary to dissect the structural basis of Atlastin function, the identification of its fusogenic properties lays the foundation for understanding the mechanisms underlying ER biogenesis and maintenance, and may contribute to a better understanding of neuronal degeneration in hereditary spastic paraplegia (Orso, 2009).

Drosophila Atlastin regulates the stability of muscle microtubules and is required for synapse development

Hereditary spastic paraplegia (HSP) is an inherited neurological disorder characterized by progressive spasticity and weakness of the lower extremities. The most common early-onset form of HSP is caused by mutations in the human gene that encodes the dynamin-family GTPase Atlastin-1 (Atl-1). Loss of the Drosophila ortholog of Atl-1 (Atl) has been found to induce locomotor impairments from the earliest adult stages, suggesting the developmental role of atlastin-subfamily GTPases. This study provides evidence that Atl is required for normal growth of muscles and synapses at the neuromuscular junction (NMJ). Atl protein is highly expressed in larval body-wall muscles. Loss-of-function mutations in the atl gene reduce the size of muscles and increase the number of synaptic boutons. Rescue of these defects is accomplished by muscular, but not neuronal expression of Atl. Loss of Atl also disrupts ER and Golgi morphogenesis in muscles and reduces the synaptic levels of the scaffold proteins Dlg and α-spectrin. Evidence is provided that Atl functions with the microtubule-severing protein Spastin to disassemble microtubules in muscles. Finally, this study demonstrates that the microtubule-destabilizing drug vinblastine alleviates synapse and muscle defects in atl mutants. Together, these results suggest that Atl controls synapse development and ER and Golgi morphogenesis by regulating microtubule stability (Lee, 2009).

During Drosophila larval development, there is a coordinated growth of the muscle and NMJ. The current results suggest that these developmental processes are impaired in the absence of Atl. In atl mutant larvae, NMJ boutons are smaller and more numerous than in wild-type larvae. In addition, the size of body-wall muscles is significantly reduced. These phenotypes are rescued by muscle-specific expression of Atl, suggesting that Atl functions primarily in the postsynaptic muscle to control NMJ synapse and muscle growth. This conclusion is further supported by the observation that targeted expression of a dominant-negative form of Atl (AtlR192Q) in muscles, but not in neurons, mimics atl mutations to increase bouton number per muscle surface area (Lee, 2009).

In Drosophila, type I NMJ boutons are surrounded by the subsynaptic reticulum (SSR), which is highly elaborated infoldings of the muscle membrane. The SSR begins to form during the first larval instar and increases in size and complexity throughout late larval life. Ultrastructural analysis indicates that the SSR is less extensive and disorganized in atl mutants. Consistent with this finding, defects were observed in the levels and distribution of Dlg and spectrin, the synaptic scaffold proteins that play critical roles for SSR expansion and organization. Thus, these results suggest that Atl is required for the formation and/or maintenance of normal postsynaptic structure (Lee, 2009).

In mammals, there are three members of the atlastin subfamily of large GTPases, each with a distinct tissue distribution. In particular, Atl-1 is primarily enriched in the CNS (Zhu, 2003), while Atl-2 and Atl-3 are highly expressed in muscle tissues (Rismanchi, 2008). This expression profile of mammalian atlastins suggests the possibility that they may play an essential role in both pre- and postsynaptic cells. In Drosophila, the single ortholog of mammalian atlastins (Atl) is expressed in the adult brain and is required for maintaining dopaminergic neurons (Lee, 2008). This study investigated the cellular function of Atl in the developing postsynaptic muscle (Lee, 2009).

In muscle cells, loss of Atl produces abnormal accumulation of stable microtubules, while its overexpression has the opposite effect. Thus, these genetic experiments reveal that Atl is involved in normal disassembly of microtubules. However, Atl is not homologous to any known microtubule-associated proteins, raising the key question as to how Atl regulates microtubule stability. Genetic and biochemical data obtained from this study and others support the model in which Atl acts through Spastin to disassemble microtubules. First, phenotypic overlap between atl and spastin is very extensive in Drosophila. atl and spastin mutants display similar adult phenotypes: short life span, impairments in locomotor activity, and age-dependent neurodegeneration (Lee, 2008; Orso, 2005; Sherwood, 2004). spastin mutations are also shown to cause an increase in bouton number and satellite bouton formation at larval NMJs (Sherwood, 2004). First, this study demonstrates that NMJ morphology is similarly affected by atl mutations. Moreover, both mutations lead to a decrease in expression of ER and Golgi markers in muscle cells. Second, this study demonstrates that Atl and Spastin can physically bind each other in vitro. Finally, it was shown that spastin-null mutation suppresses the loss of stable microtubules induced by atl overexpression (Lee, 2009).

How is Atl involved in Spastin-mediated microtubule disassembly? Proteins in the AAA family have been shown to maintain their substrate specificities often by biochemical interactions with adaptor proteins that can bring them to specific subcellular compartments or molecular targets. Potential adaptor proteins for mammalian Spastin include a centrosomal protein NA14 (Errico, 2004) and an endosomal protein CHMP1B (Reid, 2005), suggesting that Spastin may function at multiple subcellular compartments. This study shows that Atl in the Drosophila muscle localizes largely to the ER and partially to the Golgi apparatus. Together with biochemical interactions between Spastin and Atl, this result raises the possibility that Atl may function as an ER- and Golgi-specific adaptor for Spastin in the muscle. However, the possiblity cannot be excluded that Atl may modulate the microtubule-severing activity or stability of Spastin. Future work is needed to address this issue (Lee, 2009).

Dominant-negative or pathogenic forms of mammalian atlastins interfere with the formation, migration, or fusion of vesicles in the ER/Golgi interface in cultured cells (Namekawa, 2007) and disrupt the morphology of these organelles (Namekawa, 2007; Rismanchi, 2008). This study provides in vivo evidence that Drosophila Atl is required for normal formation of the ER and Golgi organelles. Thus, data obtained from mammalian cells and Drosophila support the role for Atl and its mammalian homologs in membrane trafficking from the ER and morphogenesis of the ER and Golgi apparatus. What is then the relationship between microtubule stability and morphogenesis of the ER and Golgi morphogenesis? Two lines of evidence suggest that Atl-dependent microtubule disassembly is critical for normal morphogenesis of those organelles. First, spastin-null mutants display ER and Golgi defects similar to those in atl mutants. Second, the ER and Golgi phenotype in atl is significantly rescued by administration of the microtubule-destabilizing drug vinblastine. Thus, accumulation of stable microtubules appears to be a causative mechanism to account for defects in ER and Golgi morphogenesis in atl. This conclusion is compatible with the previously reported roles for the microtubule cytoskeleton in membrane trafficking between the ER and the Golgi apparatus, as well as in the organization and distribution of those secretory organelles (Lee, 2009).

The postsynaptic defects of atl mutants are also rescued by pharmacologically reversing microtubule accumulation, raising an important question as to how the lack of Atl-mediated microtubule regulation induces abnormalities in postsynaptic structure. One possibility is that defects in vesicle trafficking induced by atl mutations could impair the delivery of synaptic membrane components, which in turn would produce an underdeveloped and disorganized SSR. If this is the case, Atl-mediated microtubule regulation may be critical for 'Golgi-bypass' secretory pathways since anterograde ER-to-Golgi protein trafficking appears to be normal in cells expressing dominant-negative forms of atlastins (Rismanchi, 2008). Such pathways include a Rab1-positive route that connects the pre-Golgi intermediate compartment (IC) directly with the cell periphery. In this regard, it will be interesting to address in the future whether synaptic components critical for SSR development are transported to the plasma membrane through the Rab1-mediated or related pathways in the muscle. An alternative and additional possibility is that loss of Atl could alter local microtubules surrounding the postsynaptic area, thereby affecting SSR structure. At the Drosophila NMJ, muscle microtubules are excluded from the peribouton area (i.e. SSR) that can be defined by the spectrin/actin network. It has been proposed that the extent of the SSR is strongly correlated with postsynaptic aPKC activity, which decreases the stability of muscle microtubules. By analogy with aPKC, Atl could support the expansion of the SSR by decreasing the stability of local microtubules surrounding the SSR. At this time, however, other mechanisms by which misregulation of muscle microtubules may alter the postsynaptic structure cannot be excluded, as observed in the postsynaptic side of atl mutant NMJs (Lee, 2009).

At present, it is still unclear how postsynaptic Atl regulates presynaptic growth. Since over-activation of retrograde BMP signaling also produces an NMJ phenotype characterized primarily by increased satellite bouton formation at the NMJ, it is an open question whether microtubule misregulation in atl inhibits the activity or the secretion from postsynaptic muscles of BMP and other retrograde growth signals (Lee, 2009).

The HSP proteins implicated in microtubule organization and/or microtubule-dependent processes include microtubule motor protein KIF5A and other molecules containing the microtubule interacting and trafficking (MIT) domain (e.g., Spartin and Spastin). Previous functional characterization of these proteins has centered on their roles in axons and presynaptic terminals. For example, Spastin has been shown to be expressed at the presynaptic compartment of the Drosophila larval NMJ, and to regulate synaptic structure and function by regulating the stability of presynaptic microtubules. However, the Spastin protein is also expressed in the postsynaptic muscle in Drosophila and humans. At present, it is unknown to what extent microtubule misregulation in the postsynaptic cell contributes to the presynaptic phenotypes observed in Drosophila spastin-null mutants and human patients with HSP SPG4. This study has clearly demonstrated in Drosophila that misregulation of muscle microtubules caused by atl mutations produces synaptic defects, highly reminiscent of those seen with spastin loss-of-function mutations. Given the biochemical and functional links between Atl and Spastin, the current work provides a new perspective on the postsynaptic role of Spastin and other microtubule-related HSP proteins during synapse development (Lee, 2009).

Loss of spastic paraplegia gene atlastin induces age-dependent death of dopaminergic neurons in Drosophila

Hereditary spastic paraplegias (HSPs) are human genetic disorders causing increased stiffness and overactive muscle reflexes in the lower extremities. atlastin is one of the major genes in which mutations result in HSP. A Drosophila model of HSP was generated that has a null mutation in atl. As they aged, atl null flies were paralyzed by mechanical shock such as bumping or vortexing. Furthermore, the flies showed age-dependent degeneration of dopaminergic neurons. These phenotypes were rescued by targeted expression of atl in dopaminergic neurons or feeding L-DOPA or SK&F 38393, an agonist of dopamine receptor. These data raised the possibility that one of the causes of HSP disease symptoms in human patients with alt mutations is malfunction or degeneration of dopaminergic neurons (Lee, 2008).


REFERENCES

Search PubMed for articles about Drosophila Atlastin

Errico, A., Claudiani, P., D'Addio, M. and Rugarli, E. I. (2004). Spastin interacts with the centrosomal protein NA14, and is enriched in the spindle pole, the midbody and the distal axon. Hum. Mol. Genet. 13: 2121-2132. PubMed ID: 15269182

Lee, M., et al. (2009). Drosophila Atlastin regulates the stability of muscle microtubules and is required for synapse development. Dev. Biol. 330(2): 250-62. PubMed ID: 19341724

Lee, Y., et al. (2008). Loss of spastic paraplegia gene atlastin induces age-dependent death of dopaminergic neurons in Drosophila. Neurobiol. Aging 29: 84-94. PubMed ID: 17030474

Namekawa, M., et al. (2007). Mutations in the SPG3A gene encoding the GTPase atlastin interfere with vesicle trafficking in the ER/Golgi interface and Golgi morphogenesis. Mol. Cell. Neurosci. 35: 1-13. PubMed ID: 17321752

Orso, G. et al. (2005). Disease-related phenotypes in a Drosophila model of hereditary spastic paraplegia are ameliorated by treatment with vinblastine. J. Clin. Invest. 115: 3026-3034. PubMed ID: 16276413

Orso, G., et al. (2009). Homotypic fusion of ER membranes requires the dynamin-like GTPase atlastin. Nature 460(7258): 978-83. PubMed ID: 19633650

Praefcke, G. J. and McMahon, H. T. (2004). The dynamin superfamily: universal membrane tubulation and fission molecules? Nature Rev. Mol. Cell Biol. 5: 133-147. PubMed ID: 15040446

Reid, E., et al. (2005). The hereditary spastic paraplegia protein spastin interacts with the ESCRT-III complex-associated endosomal protein CHMP1B. Hum. Mol. Genet. 14: 19-38

Rismanchi, N., Soderblom, C., Stadler, J., Zhu, P. P. and Blackstone, C. (2008). Atlastin GTPases are required for Golgi apparatus and ER morphogenesis. Hum. Mol. Genet. 17: 1591-1604. PubMed ID: 18270207

Sanderson, C. M., et al. (2006). Spastin and atlastin, two proteins mutated in autosomal-dominant hereditary spastic paraplegia, are binding partners. Hum. Mol. Genet. 15: 307-318. PubMed ID: 16339213

Sherwood, N. T., et al. (2004). Drosophila spastin regulates synaptic microtubule networks and is required for normal motor function. PLoS Biol. 2: e429. PubMed ID: 15562320

Zhao, X. et al. (2001). Mutations in a newly identified GTPase gene cause autosomal dominant hereditary spastic paraplegia. Nature Genet. 29: 326-331. PubMed ID: 11685207

Zhu, P. P., et al. (2003). Cellular localization, oligomerization, and membrane association of the hereditary spastic paraplegia 3A (SPG3A) protein atlastin. J. Biol. Chem. 278: 49063-49071. PubMed ID: 14506257


Biological Overview

date revised: 20 February 2010

Home page: The Interactive Fly © 2009 Thomas Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.