InteractiveFly: GeneBrief

atlastin: Biological Overview | References

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 (Atl^R192Q) 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).

The effects of ER morphology on synaptic structure and function in Drosophila melanogaster

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 (Summerville, 2016).

The function of intracellular organelles is tightly coordinated with location within the cytoplasm. The endoplasmic reticulum (ER) is an interconnected network of narrow tubes and flattened cisternae or sheets. In most cells, the ER is the most abundant subcellular organelle and extends elaborate processes throughout the cytoplasm. The ER membrane is formed into its tubular architecture by the action of structural proteins within the reticulon, REEP and DP1 family. The members of this diverse family of proteins share a common protein motif called the reticulon homology domain (RHD). The hydrophobic ~200-amino-acid RHD likely forms a helical hairpin structure that intercalates four hydrophobic helical segments into the outer leaflet of the ER membrane to induce curvature and maintain a tubular shape. Many members of the Reticulon, REEP and DP1 family also contain an extended N-terminal segment ranging from a few hundred to a thousand amino acids that likely provides additional functionality. The nature of most of these secondary functions remains to be revealed (Summerville, 2016).

The large ER network also maintains luminal and membrane continuity throughout the cytoplasm. This interconnected nature of the ER network is required for ER function and is maintained by the ER membrane fusion GTPase atlastin, which is a member of the fusion dynamin-related protein family (fusion DRP)(Summerville, 2016).

The ER is closely associated with and functionally connected to the plasma membrane. This connection is often associated with the management of Ca2+ stores in the ER lumen. The ER protein STIM1 diffuses through the ER membrane to find binding partners in the plasma membrane including the Orai channel. This set of protein-protein associations works to restore ER Ca2+ through the store-operated Ca2+ channel system. ER-plasma-membrane contact sites are also generated by the association of ER-integral extended synaptotagmins (E-Syt) with phospholipids in the plasma membrane as well as proteins like junctophillins in certain cell types (Summerville, 2016).

Most recently, the ER has been found to be stably associated with endosomal structures. In this circumstance, the specific proteins on each surface that interact remain to be precisely defined, but the consequence of the interaction is functional segregation of certain cargoes within the endosome that permits regulated sorting into membrane subdomains prior to an ER-directed membrane fission event (Summerville, 2016).

ER structure appears to be crucially important for cell function given that human disease results when components that control this structure are compromised by mutation. The hereditary spastic paraplegias (HSPs) are a group of related genetic disorders caused by mutations in any of more than 70 genes, denoted SPG1 to SPG72. Lower limb weakness and spasticity represent two prominent clinical features of these diseases, which occur as a consequence of dysfunction or degeneration of the upper motor neurons. The observation that atlastin 1 (ATL1) and reticulon 2 (RTN2) are HSP genes responsible for SPG3A and SPG12, respectively, implicates ER morphology in the neuronal dysfunction that causes HSPs (Summerville, 2016).

The properties of three additional HSP genes, spartin (SPG20), spastin (SPAST, also known as SPG4) and NIPA1 (also known as spichthyin or SPG6), implicate receptor trafficking through the endocytic system in HSP neuronal dysfunction. For example, loss of spartin attenuates both ligand-stimulated EGF receptor uptake as well as depolarization-stimulated FM1-43 uptake, whereas loss of spastin increases endosome tubule number and alters transferrin receptor sorting). NIPA1 is also located in endosomes and promotes the endocytosis of receptors for bone morphogenetic protein (BMP) (Summerville, 2016).

Phenotypic analysis of mutations in the HSP orthologs of model systems has provided additional clues to the cellular function of these proteins. In Drosophila, loss of spastin, spartin and spichthyin confers a similar, but not identical, set of phenotypes including stabilized microtubules, increased synaptic bouton number and decreased evoked transmitter release at the larval neuromuscular junction (NMJ), age-dependent locomotor deficits and increased BMP signaling at the larval NMJ. These shared phenotypes might reflect disruption of a common pathway in endocytic receptor trafficking in these mutants. Some of these phenotypes, such as stabilized microtubules, age-dependent locomotor deficits and increased synaptic bouton number, are also observed in flies lacking atlastin (atl) (Summerville, 2016).

This study extends this phenotypic analysis of altered atl and reticulon-like 1 (Rtnl1) activities in Drosophila. The ER in motor nerve terminals from wild-type larvae forms a network of tubules that resembles a 'basket', but is diffuse in larvae lacking atl. Neuronal RNA interference (RNAi)-mediated knockdown of either atl or Rtnl1 increases arborization at the larval neuromuscular junction and decreases evoked transmitter release from larval motor nerve terminals, and elevated bath [Ca2+] fully rescues these transmitter release phenotypes. This study also showed that atl is required only in motor neurons to affect transmitter release, whereas Rtnl1 is required additionally in the target muscle and peripheral glia. This study shows that loss of atl increases BMP signaling in larval motor neurons and causes age-dependent locomotor deficits in adults. Thus, loss of atl and Rtnl1 confers phenotypes similar, but not identical, to each other as well as to mutants defective in spartin, spastin and spichthyin. These results demonstrate specific mechanistic links between ER morphology and several aspects of neuronal anatomy and function (Summerville, 2016).

Mutations in two genes that affect ER morphology, atlastin (ATL1) and reticulon 2 (RTN2), cause two forms of hereditary spastic paraplegia (HSP), which result in progressive limb weakness, spasticity and degeneration of the longest motor axons. These observations suggest that altered ER morphology is causal for motor axon dysfunction, but the mechanisms underlying these dysfunctions are unclear. This study used Drosophila to evaluate the nervous system deficits caused by altered atl and Rtnl1 activity. Using a new fluorescent ER imaging reagent, it was shown that the ER in wild-type motor nerve terminals is present as a network of tubules, termed 'baskets', underlying the plasma membrane, and that these baskets are eliminated in larvae lacking or overexpressing atl. This study also shows that loss of either atl or Rtnl1 increases arborization and decreases evoked transmitter release, and that evoked release is restored to normal by elevated bath [Ca2+]. Finally, atl loss was shown to increase signaling through the bone morphogenetic protein (BMP) pathway and causes age-dependent declines in adult locomotion. This set of phenotypes is also exhibited by Drosophila mutant for the HSP orthologs of spartin, spastin and spichthyin, as well as for spinster and nervous wreck, which encode regulators of receptor trafficking through endosomes (Summerville, 2016).

Two adjustments were made to improve visualization of the ER. First, a transgene was introduced into flies that encoded an ER-localized superfolder GFP, which was optimized for efficient folding in the ER. Second, based on previous results indicating that the ER as well as the lysosomal tubule network is labile to fixation, ER was imaged in live tissues. Using these approaches, the ER was shown to be present within the axon initial segments of motor neurons as a polygonal structure with numerous crossbridges (three-way junctions), and in motor nerve terminals as a network of tubules that were termed 'baskets', which underlie the plasma membrane. It was also shown that these structures are disrupted by either loss of or overexpression of atl. In particular, atl overexpression causes the aberrant appearance of large punctae in motor neuron cell bodies or axon initial segments. In contrast, atl loss decreases the number of crossbridges in the axon initial segment, leading to excessively long tubules. A similar appearance was noted previously and attributed to deficits in fusion of orthogonal ER membranes. Loss of atl also disrupts nerve terminal baskets and appears to cause ER fragmentation. It is possible that the transition from tubules to baskets as the ER moves from the interbouton region to boutons occurs through ER fragmentation followed by Atl-dependent reassembly. In this view, loss of atl would prevent this reassembly, thus causing the fragmented ER that was observed (Summerville, 2016).

Evoked transmitter release deficits in both atl² and Rtnl1¹ mutants, and in pan-neuronal atl or Rtnl1 knockdown larvae, were rescued partially or completely by elevated bath [Ca2+]. These results indicate that loss of atl or Rtnl1 decreases evoked transmitter release at low bath [Ca2+] through causing deficits in evoked increases in cytoplasmic [Ca2+]. Insufficient Ca2+ influx could result from attenuated action potentials, which would decrease the opening of voltage-gated Ca2+ channels, decreases in number of plasma membrane Ca2+ channels or decreased Ca2+ release from the ER. Given the role of atlastin and the reticulons as ER-shaping molecules, effects on ER Ca2+ release would be the most direct explanation for this Ca2+ phenotype. ER-localized Ca2+ release channels such as the inositol 1,4,5-trisphosphate (IP3) receptor, the ryanodine receptor and the TRPV1 channel play key roles in evoked neurotransmitter release. In addition, dominant-negative mutations in the Drosophila ER-localized Ca2+ pump SERCA decrease evoked transmitter release by ~50%, which is consistent with the possibility that ER-derived Ca2+ contributes significantly to the Ca2+ required to trigger transmitter release (Summerville, 2016).

Unlike Atl, which appears to affect evoked transmitter release from neurons alone, Rtnl1 is required in neurons, muscles and peripheral glia for correct evoked transmitter release. This finding is consistent with previous data demonstrating that proper synaptic transmission requires intercellular signaling among these three cell types. In particular, loss of activity within the peripheral glia of the kinesin heavy chain gene or the inebriated-encoded neurotransmitter transporter alters evoked transmitter release. In addition, the peripheral glia secrete at least two proteins, the TGF-β ligand Maverick and Wingless/Wnt, that regulate synaptic function. The muscle, in turn, secretes the BMP ligand Gbb to regulate both evoked transmitter release and motor neuron arborization. It is possible that loss of Rtnl1 affects transmitter release from glia or muscle by perturbing secretion of these or other regulators (Summerville, 2016).

The most prominent clinical symptom in HSP patients is progressive, age-dependent locomotor difficulties. Drosophila mutant for any of several HSP orthologs, including spartin, spastin, atl and Rtnl1, as well as spinster, exhibit similar age-dependent locomotor deficits or lifespan deficits. This study shows locomotor impairment in adults with neuronal-specific atl knockdown. These results indicate a requirement for atl in neurons for proper locomotion but do not rule out crucial roles for atl in other tissues as well (Summerville, 2016).

Mutants in Drosophila orthologs of several HSP genes, including spartin, spastin and spichthyin, and the additional related genes spinster and nervous wreck share a common set of nervous system phenotypes, including increased arborization and BMP signaling at the larval NMJ, decreased evoked transmitter release and locomotor deficits (note that not all phenotypes have been reported for each mutant). The encoded proteins localize to various compartments within the endocytic receptor trafficking pathway. In fact, the increased BMP signaling in several of these mutants has been attributed to trafficking defects of the BMP receptor Wishful thinking (Wit). This study has shown that atl loss confers these same phenotypes, raising the possibility that atl acts in the endocytic receptor trafficking pathway as well. Although the ER is not known to play prominent roles in this pathway, a recent report has demonstrated that the ER is required for endosome fission in COS cells, and, in fact, the ER selects the location of fission. In addition, it has been found that this process is inhibited by overexpression of Rtn4a, which, similarly to atl loss, elongates ER tubules and inhibits formation of crossbridges. Thus, loss of atl could impact on the receptor trafficking pathway in Drosophila nerve terminals by similarly preventing endosome fission (Summerville, 2016).

The variety of phenotypes exhibited in common by the mutants described above raises the possibility that certain phenotypes might have causal relationships with others. The subcellular locations of these proteins suggest that they might directly affect receptor trafficking. If so, then the increased BMP signaling, as a consequence of altered Wit trafficking, might be the direct cause of the increased arborization and locomotor deficits. The phenotypes conferred by direct activation of the BMP pathway in neurons are consistent with this possibility. However, the increased BMP signaling is unlikely to cause the decreased transmitter release, as decreased BMP signaling, rather than increased BMP signaling, decreases evoked transmitter release. It is suggested that trafficking of receptors in addition to Wit are altered in the mutants described above, and it is the altered signaling of these additional receptors that is at least partly responsible for the transmitter release phenotype. Drosophila motor nerve terminals express a cholecystokinin-like receptor (CCKLR), a toll-like receptor, a metabotropic glutamate receptor (mGluRA) and likely the insulin receptor. Loss of mGluRA increases evoked transmitter release, raising the possibility that increased mGluRA signaling might decrease transmitter release, which could explain the decreased transmitter release observed in these receptor trafficking mutants (Summerville, 2016).

Spastin, atlastin and ER relocalization are involved in axon, but not dendrite, regeneration

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 (Rao, 2016).

Previous work has shown that axon regeneration is impaired when one copy of spastin is mutant. This study now shows that atlastin is also haploinsufficient for axon regeneration and that reduction of several other HSP proteins using RNAi impairs regeneration. Thus axon regeneration seems to be a postdevelopmental process that involves at minimum a subset of HSP protein (Rao, 2016).

The sensitivity of axon regeneration to partial reduction of HSP proteins, however, depends on the genetic background. In a previous study, EB1-GFP was used as a dual-purpose marker of cell shape and microtubule dynamics. This fusion protein is not, however, completely neutral. GFP fused to the C-terminus of EB1 can interfere with binding of partner proteins to EB1. Because EB1 acts as a dynamic platform at growing microtubule ends that recruits other proteins, the presence of large amounts of EB1-GFP could reduce recruitment of other plus end-binding proteins. Indeed, in Drosophila, neurons EB1 binds Apc, which in turn brings kinesin-2 to growing dendritic microtubules to help maintain minus-end-out polarity, and high levels of EB1-GFP result in mixed polarity. Because of this, this study expressed EB1-GFP at low levels, but it is still possible that there is a subtle defect in microtubule growth or organization. Under normal circumstances, this does not result in any defects in regeneration, which is indistinguishable in control neurons expressing EB1-GFP, mCD8-RFP, or Rtnl1-GFP. Only when combined with partial reduction of HSP proteins was a difference seen among neurons expressing different markers. This difference was most clearly demonstrated in spastin heterozygotes, which had a very strong reduction in regenerative growth in neurons labeled with EB1-GFP but not with mCD8-RFP. The synthetic interaction between EB1-GFP and spastin suggests that even though the early microtubule changes triggered by axon injury were normal, with reduced levels of HSP proteins, microtubules were somehow involved in the phenotype. This conclusion was supported by a similar effect of EB1-CT, a dominant-negative form of EB1, and the fact that introduction of tdEOS-αtubulin suppressed the spastin phenotype (Rao, 2016).

To probe in more depth how HSP protein function related to regenerative axon growth, several other approaches were taken. First, dendrite regeneration was examined. Complete regeneration of dendrites after removal of the entire arbor involves very rapid outgrowth, and so it was reasoned that if HSP proteins were generally involved in facilitating growth of neuronal processes, they should be required for dendrite regeneration. No defects in dendrite regeneration were observed, and this suggested that the cells were healthy and that a process specific to axon regeneration was sensitive to HSP protein reduction. Second, a catalogue of intracellular markers was examined to look for rearrangements associated with regenerative axon growth. The ER was most dramatically different in regenerating axons and accumulated near the growing tip. Moreover, this was specific to axon regeneration and not observed in dendrite regeneration (Rao, 2016).

All of these observations were assembled into a model for HSP protein function during regeneration. It is proposed that a subset of HSP proteins is involved in concentrating the ER, together with underlying microtubules, at tips of axons undergoing regenerative growth. This model makes particular sense for spastin and atlastin. Because spastin is a microtubule regulator that in flies and mammals also has a transmembrane domain and binds the ER regulatory protein atlastin, their combined action could facilitate concentration of the ER at growing axon tips. In support of this idea, reduction of either protein disrupted ER accumulation at single growing axon tips (Rao, 2016).

Although it is believed that atlastin and spastin help to concentrate ER at growing axon tips by linking the ER to microtubules, which also accumulate at growing tips, it is not known what mediates the microtubule redistribution. It is suspected, however, that microtubule polarity is involved in setting up tip accumulation of tubulin and ER. Tip accumulation is seen only during regenerative axon growth, which requires microtubules in the growing process to be plus-end-out, and not during regenerative dendrite growth, when microtubules are largely minus-end-out. In initiation of regenerative axon outgrowth in cultured Drosophila neurons, kinesin-mediated microtubule sliding is important, and so one possibility is that motors slide short pieces of microtubules out to the new tip (Rao, 2016).

It is intriguing that ER accumulates at regenerating axon but not dendrite tips. This suggests that increased amounts of local ER are specifically important for promoting maximal axon growth. Local Smooth Endoplasmic Reticulum (SER) could promote axon growth by increasing local lipid production or increasing availability of intracellular calcium. A recent study in Caenorhabditis elegans suggests that it is the calcium storage function of SER that is important in this context. In this system, release of ER calcium through ryanodine receptors is required for maximal axon outgrowth, and high levels of calcium were seen at axon tips up to 5 h (the latest time point examined) after axon injury. Thus perhaps atlastin and spastin help concentrate ER at growing axon tips to provide a local source of intracellular calcium stores, which in turn facilitate regenerative growth (Rao, 2016).

It is difficult to know how a function for atlastin and spastin, and potentially other HSP proteins, in ER localization during regenerative axon growth relates to the axon degeneration that occurs in the disease. All HSP proteins seem to have important basic cellular functions that are quite universal. For example, atlastin is biochemically an ER fusion protein, but only a subset of disease-causing atlastin mutations affect ER fusion. Perhaps the function of atlastin, spastin, and other HSP proteins important for disease is not the core function but a subtler role these proteins play in very long neurons. ER relocalization during regenerative growth of axons is worth considering as a disease-relevant function for several reasons: 1) it is important for mature neurons, 2) at least two different HSP proteins contribute to it, 3) repeated small failures of regeneration could lead to accumulated axon loss over a long time period, and 4) at least in some genetic backgrounds, the capacity for regenerative growth is reduced when only one allele of the gene is mutant. An interesting further speculation is how this function might relate to cell-type susceptibility to regeneration. The hallmark of HSP is degeneration of upper motor neurons. As in many neurodegenerative diseases, it is unclear why these cells might be more sensitive than others. One possibility raised by the data is that the suite of microtubule regulators expressed in different neurons could influence sensitivity of regeneration to reduction of HSP proteins. For example, slightly higher levels of a microtubule-stabilizing protein might eliminate the need for full HSP protein function during regeneration in the same way that tdEOS-αtubulin bypassed the requirement for spastin and atlastin for regeneration. Similarly, a different set or ration of microtubule plus end-binding proteins could make a particular neuron type more sensitive to partial reduction of HSP proteins in the same way that EB1-GFP and EB1-CT did in this study (Rao, 2016).

Although the idea that the function identified for HSP proteins during axon regeneration is appealing in many ways, it is not a perfect fit. It is not known whether axon regeneration is triggered during normal wear and tear of axons in the spinal cord, and this is a critical missing piece of information necessary to evaluate whether reduction in regeneration might lead to long-term degeneration (Rao, 2016).

Independently of potential relevance to disease, the differential effect of reduction of HSP proteins on axon and dendrite regeneration is intriguing. One might expect that proteins with core cellular functions like ER and microtubule regulation would be equally required for both types of outgrowth. Similarly, if the ER is concentrated at growing axon tips to provide an extra calcium reservoir, why is this not important for dendrite regenerative growth? It will be extremely interesting to learn what promotes dendrite regeneration in future studies (Rao, 2016).

Dynamic constriction and fission of endoplasmic reticulum membranes by reticulon

The endoplasmic reticulum (ER) is a continuous cell-wide membrane network. Network formation has been associated with proteins producing membrane curvature and fusion, such as reticulons and atlastin. Regulated network fragmentation, occurring in different physiological contexts, is less understood. This study finds that the ER has an embedded fragmentation mechanism based upon the ability of reticulon to produce fission of elongating network branches. In Drosophila, Rtnl1-facilitated fission is counterbalanced by atlastin-driven fusion, with the prevalence of Rtnl1 leading to ER fragmentation. Ectopic expression of Drosophila reticulon in COS-7 cells reveals individual fission events in dynamic ER tubules. Consistently, in vitro analyses show that reticulon produces velocity-dependent constriction of lipid nanotubes leading to stochastic fission via a hemifission mechanism. Fission occurs at elongation rates and pulling force ranges intrinsic to the ER, thus suggesting a principle whereby the dynamic balance between fusion and fission controlling organelle morphology depends on membrane motility (Espadas, 2019).

The endoplasmic reticulum (ER) comprises two uninterrupted domains, the nuclear envelope and the peripheral ER. The peripheral ER is composed of structural elements with different membrane curvature and topology, from flat sheets and reticulated tubules to complex fenestrated structures. These elements are distributed throughout the cytoplasm of the eukaryotic cell as a membrane network enclosing a single lumen. Network maintenance requires homotypic membrane fusion mediated by the atlastin family of dynamin-related GTPases. Suppression of atlastin fusogenic activity leads to ER fragmentation, thus revealing an endogenous mechanism aimed at the reduction of ER connectedness. The existence of this mechanism has been confirmed by several reports showing ER disassembly during mitosis, reversible fragmentation of the ER both in neurons and other cell types, and fragmentation of the ER prior to autophagic degradation. Furthermore, fission of individual ER branches was recently detected by using super-resolution live-cell imaging of the ER network. While no dedicated molecular machinery has been linked to ER fragmentation, few experimental observations suggest an involvement of reticulons, highly conserved integral ER membrane proteins implicated in shaping and stabilizing the tubular ER1. Notably, mutations in both Reticulon-2 and Atlastin-1 have been linked to the neurodegenerative disorder hereditary spastic paraplegia, corroborating their participation in coordinated functional and pathological pathways (Espadas, 2019).

Overexpression of members of the Yop1 and reticulon families of proteins has been reported to cause severe constriction of ER branches and ER fragmentation. Fragmentation could proceed via the breakage of ER tubules, implicating high local curvature stress and membrane fission. Fragmentation was also linked to the shedding of small vesicles, a process whose significance in ER fragmentation, however, is not understood. Tubule fission would naturally antagonize the fusogenic activity of atlastin in the ER, making fusion/fission balance a paradigm in intracellular organelle maintenance. Despite the reported association between reticulons and ER fragmentation, direct involvement of reticulons has not been shown and the mechanism(s) of fragmentation remains obscure. Furthermore, creation of membrane curvature by reticulons was mechanistically linked to construction, not fragmentation of the tubular ER network, both in vitro and in vivo. In agreement with involvement in formation rather than fragmentation of the tubular ER, purified reticulons reconstituted into lipid vesicles induced membrane curvatures insufficient to produce membrane fission (Espadas, 2019).

This study reveals the mechanism underlying reticulon membrane activity that unifies these seemingly contradictory observations. rosophila Reticulon (Rtnl1), while promoting ER tubulation and enhancing the total curvature of ER membranes, is also responsible for ER fragmentation via membrane fission. Fragmentation occurs both at endogenous levels of Rtnl1, when unchallenged due to the absence of atlastin, and upon Rtnl1 overexpression. Corroborating these in vivo results, purified Rtnl1 reconstituted into dynamic lipid nanotubes produces curvatures ranging from moderate, as reported earlier (Hu, 2008), to those causing spontaneous membrane fission. In vivo, this ability of Rtnl1 to induce membrane fission is counterbalanced by atlastin, with the interplay between these proteins exerting the core control on total curvature and connectedness of the ER network in a living organism (Espadas, 2019).

Ever since the discovery of homotypic fusion of ER membranes by atlastin there have been indications in the literature of the existence of an endogenous mechanism balancing unceasing fusion during ER network maintenance. Recent studies, both in vitro and in vivo, reiterated the physiological importance of ER fragmentation and linked it to the curvature-creating proteins operating in the ER15,49. Yet, the puzzle remained as to how proteins implicated in making the tubular ER network, such as reticulons, could also mediate fragmentation of the same network. The results demonstrate that these seemingly opposite functions can indeed exist in a single protein, Rtnl1, combining two different modes of curvature creation, static, and dynamic. The static mode, associated with local membrane bending by the membrane-inserting domains of reticulons, accounts for mechanical stabilization of membrane tubes. The dynamic mode, associated in this work with the increased viscosity of Rtnl1-containing membranes, accounts for friction-driven constriction of elongating membrane tubules, leading to their scission. Dynamic coupling between these two modes via curvature-driven sorting of Rtnl1 toward the nanotube is absolutely critical for fission to occur. Viscous drag alone would produce nanotube constriction only at elevated tensile stress and thus result in the mechanical rupture of the membrane. Dynamic accumulation of Rtnl1 in the curved nanotubes, however, critically amplifies constriction so that scission can happen at reduced tensile stress, via a hemi-fission mechanism. Thus, the hemi-fission curvature threshold can be reached at physiological elongation, speeds and forces, within a range of Rtnl1 concentration that creates only the moderate static curvatures required for ER tubule stabilization. Hence, in the dynamic ER network Rtnl1 readily combines its membrane curvature stabilization and fission activities without risking the leakage of the ER lumen contents into the cytoplasm (Espadas, 2019).

In ER maintenance, membrane fission by Rtnl1 must be balanced by atlastin-mediated membrane fusion. Fundamentally, this balance is described by a kinetic model which explicitly accounts for the two opposing functions of Rtnl1, static curvature stabilization and dynamic fission. The intrinsic sensitivity to membrane dynamics suggests a paradigm of dynamic regulation of ER topology linking membrane fusion and fission with membrane motility. This paradigm implies that ER fragmentation, a process crucial in physiological conditions, for example maintenance of ER morphology and ER-phagy, and likely involved in neuropathological processes can be implicitly controlled by multiple factors connected to ER motility and stresses, with Rtnl1 constituting the core element of the ER-specific membrane fission machinery (Espadas, 2019).

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).

Reconstitution of human atlastin fusion activity reveals autoinhibition by the C terminus

ER network formation depends on membrane fusion by the atlastin (ATL) GTPase. In humans, three paralogs are differentially expressed with divergent N- and C-terminal extensions, but their respective roles remain unknown. This is partly because, unlike Drosophila ATL, the fusion activity of human ATLs has not been reconstituted. This study reports successful reconstitution of fusion activity by the human ATLs. Unexpectedly, the major splice isoforms of ATL1 and ATL2 are each autoinhibited, albeit to differing degrees. For the more strongly inhibited ATL2, autoinhibition mapped to a C-terminal α-helix is predicted to be continuous with an amphipathic helix required for fusion. Charge reversal of residues in the inhibitory domain strongly activated its fusion activity, and overexpression of this disinhibited version caused ER collapse. Neurons express an ATL2 splice isoform whose sequence differs in the inhibitory domain, and this form showed full fusion activity. These findings reveal autoinhibition and alternate splicing as regulators of atlastin-mediated ER fusion (Crosby, 2022).

Search PubMed for articles about Drosophila Atlastin

Crosby, D., Mikolaj, M. R., Nyenhuis, S. B., Bryce, S., Hinshaw, J. E. and Lee, T. H. (2022). Reconstitution of human atlastin fusion activity reveals autoinhibition by the C terminus. J Cell Biol 221(2). PubMed ID: 34817557

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

Espadas, J., Pendin, D., Bocanegra, R., Escalada, A., Misticoni, G., Trevisan, T., Velasco Del Olmo, A., Montagna, A., Bova, S., Ibarra, B., Kuzmin, P. I., Bashkirov, P. V., Shnyrova, A. V., Frolov, V. A. and Daga, A. (2019). Dynamic constriction and fission of endoplasmic reticulum membranes by reticulon. Nat Commun 10(1): 5327. PubMed ID: 31757972

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

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

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

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 129(8):1635-48. PubMed ID: 26906425

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

date revised: 1 March 2024

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