InteractiveFly: GeneBrief

Reticulon-like1: Biological Overview | References


Gene name - Reticulon-like1

Synonyms -

Cytological map position - 25B9-25C1

Function - transmembrane protein that promotes membrane curvature

Keywords - a reticulon family member, with intramembrane hairpin domains that insert into the cytosolic face of the endoplasmic reticulum (ER) thereby curving it and promoting ER tubule formation - enriched on tubular ER, including axons and egg chamber fusomes, high level leads to ER fragmentation, microtubule cytoskeleton is involved in Rtnl1 localization to spindles during mitosis

Symbol - Rtnl1

FlyBase ID: FBgn0053113

Genetic map position - chr2L:4,992,808-4,999,502

Classification - Reticulon

Cellular location - ER transmembrane



NCBI links: EntrezGene, Nucleotide, Protein
BIOLOGICAL OVERVIEW

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).

Microtubules are necessary for proper Reticulon localization during mitosis

During mitosis, the structure of the Endoplasmic Reticulum (ER) displays a dramatic reorganization and remodeling, however, the mechanism driving these changes is poorly understood. Hairpin-containing ER transmembrane proteins that stabilize ER tubules have been identified as possible factors to promote these drastic changes in ER morphology. Recently, the Reticulon and REEP family of ER shaping proteins have been shown to heavily influence ER morphology by driving the formation of ER tubules, which are known for their close proximity with microtubules. This study examine the role of microtubules and other cytoskeletal factors in the dynamics of a Drosophila Reticulon, Reticulon-like 1 (Rtnl1), localization to spindle poles during mitosis in the early embryo. At prometaphase, Rtnl1 is enriched to spindle poles just prior to the ER retention motif KDEL, suggesting a possible recruitment role for Rtnl1 in the bulk localization of ER to spindle poles. Using image analysis-based methods and precise temporal injections of cytoskeletal inhibitors in the early syncytial Drosophila embryo, this study shows that microtubules are necessary for proper Rtnl1 localization to spindles during mitosis. Lastly, it was shown that astral microtubules, not microfilaments, are necessary for proper Rtnl1 localization to spindle poles, and is largely independent of the minus-end directed motor protein dynein. This work highlights the role of the microtubule cytoskeleton in Rtnl1 localization to spindles during mitosis and sheds light on a pathway towards inheritance of this major organelle (Diaz, 2019).

Research over the last decade has highlighted the dramatic changes of the ER during cell division, however the factors that govern these mitotic changes are poorly understood. This study has focused on the dynamics of the highly conserved ER shaping protein, Rtnl1, during mitosis. Rtnl1 displays a steady enrichment at the nuclear envelope and spindle poles prior to bulk of the ER membrane at prometaphase. Using precise temporal inhibition, this study shows that microtubule dynamics are necessary for proper ER localization and partitioning during mitosis. Furthermore, the small molecule inhibitors, Binucleine 2 and BI 2536, which affect the formation of astral microtubules, leads to defects in ER localization at the spindle poles early in mitosis. Blocking cytoplasmic dynein both by small molecule injection and RNAi does not affect localization of Rtnl1 at the poles. This work highlights the mechanistic requirements of mitotic ER localization and provides a framework for ER partitioning during cell division (Diaz, 2019).

A general concern with the approach of using small molecule inhibitors to examine mitotic ER organization is that they can be broad acting and the defects observed can be attributed to indirect or downstream disruptions of the cell cycle. However, it is believed that the phenotypes observed for Rtnl1 are direct outcomes of disruption of the cytoskeletal networks. This is in large part to a prior study that showed when the cell cycle was arrested using small molecule inhibitors either in interphase or mitosis, ER structure initially was unaffected and only after 15-20 minutes of arrest, were any ER defects observed. The ER defects shown in this study were immediate, within 1-2 minutes after exposure to the small molecule indicating a more direct role (Diaz, 2019).

Research over the last decade has elucidated that the ER is a dynamic organelle drastically changing its shape and localization upon entry into mitosis. The mitotic factors responsible for these dramatic changes involving the ER remains an area of great interest. It has been well established that organization of the ER relies on the microtubule network during interphase allowing the ER to stretch from the nuclear envelope to the cell periphery, however it is unknown if the microtubule network performs a similar organizational role during mitosis. Studies involving ER movement during mitosis in S. cerevisiae and C. elegans have implicated the actin cytoskeleton network in mitotic ER dynamics. The ER shares a close localization with the mitotic spindle and poles and it has generally been thought that microtubules and associated motor proteins are responsible for mitotic ER organization and partitioning. To this end, an investigation into the ER transmembrane protein, STIM1 showed an interaction with the microtubule plus-end tracking protein (+TIP) EB1. Additionally, this interaction between the ER and microtubule network is regulated by phosphorylation of STIM1. However, sequence analysis between the mammalian STIM1 and the isoforms of the Drosophila homolog, dSTIM1, showed that dSTIM1 does not contain the identified EB1 binding site or the serine or threonine regulatory amino acids, indicating the existence of multiple mechanisms for ER / microtubule interactions. In support of the role of microtubules, there have been studies implicating the astral microtubule network in partitioning of the ER during asymmetric neuroblast divisions (Diaz, 2019).

The data display a strong localization of ER at the spindle poles during mitosis (Bergman, 2015; Bobinnec, 2003). It has been assumed that ER is transported towards the poles by the major cellular minus-end directed motor, dynein. In support of this, cytoplasmic dynein has been implicated in trafficking and transport through the secretory system, as well as in the structural support and localization of organelles during interphase. It is also well established that dynein is involved in several mitotic processes including nuclear envelope breakdown through pulling forces along the astral microtubule network, thereby leading to bipolar spindle formation at metaphase. In addition, dynein has also been implicated in the transport and localization of recycling endosomes at the spindle poles during early mitosis. This study shows that small molecule inhibition of dynein during mitosis, while disrupting proper spindle assembly, did not prevent ER localization at the spindle poles. This qualitative result of dynein independence is in line with a very recent study showing that dynein does not affect ER movement to the spindle poles in Drosophila spermatocytes. This result suggests that ER is not being transported or maintained at the minus-end of the microtubules by dynein. However, the quantitative approach carried out in this study suggests that a small amount of Rtnl1 movement is affected and largely lags along the nuclear envelope. While this lack of movement was shown to be significant, this can be explained by the known role of dynein involvement in the breakdown of the nuclear envelope thereby affecting timing of NEB and release of the mitotic kinase Cyclin A affecting ER reorganization rather than a direct connection of dynein with Rtnl1 (Diaz, 2019).

Gurel (2014), in a review focused on ER calcium sequestration suggested two models of microtubule-based ER transport, the sliding mechanism and / or the tip attachment complex (TAC). Sliding mechanism focuses on motor-based movement along an existing microtubule, while TAC model suggest that ER structural proteins would attach to a +TIP protein and movement would be connected to microtubule growth. While there is evidence in different systems for each, the current data suggest that there is a direct connection to microtubule dynamics of growth and stability of the ER. Furthermore, based on small molecule inhibition of the astral microtubule network, this suggests that astral microtubule dynamics also are key in proper mitotic localization of ER at the poles. Recently, a study also showed that the kinesin-14 family member of microtubule minus-end directed motor protein, non-claret disjunctional (ncd) or the kinesin 5 plus-end directed microtubule motor protein Klp61F did not affect ER movement to the spindle poles. Future studies should investigate other possible candidate proteins including the astral microtubule associated kinesin motor protein Khc-73 or the NUMA ortholog, Mushroom body defective (Mud) that associate with the spindle poles or along the astral microtubule network (Diaz, 2019).

Early investigations into the mechanism that regulates ER shape and structure identified a class of proteins, known as Reticulons. This protein family is not defined by sequence homology, but rather by the presence of two short hairpin transmembrane domains on the cytoplasmic leaflet of the ER (Pendin, 2011). Recent studies have begun to elucidate the role of these Reticulon family members in both regulating the structural changes of the ER and connection to the cytoskeleton (Schlaitz, 2013; Pendin, 2011). Rtnl1 was the first reticulon family member identified in Drosophila, however several additional proteins with reticulon homology domains (RHD) have recently been identified in Drosophila and other systems including spastin, atlastin-1, DP1/YOP and REEPs. It has been shown that these proteins can oligomerize and form homomeric and heteromeric complexes in regards to ER tubule formation (Shibata, 2008). Recently, the mammalian REEP3/4 proteins have been shown to contribute to membrane curvature changes in mitosis and interact with microtubules. However, it is unclear if the REEP proteins work in concert with the reticulons during mitosis. The current data demonstrate the importance of the microtubule network in organizing mitotic ER. Furthermore, it is believed that the disruption of the mitotic spindle and ER organization is a direct outcome and not a downstream consequence of affecting mitotic progression, as a previous study in the early embryo showed that ER dynamics are in frame with the cell cycle and were halted when cell cycle was blocked. However, even with these disruptions, the ER maintained its mitotic organization. Future directions regarding mitotic ER organization would be to identify additional targets that regulate ER organization and partitioning during mitosis. An interesting candidate is the microtubule severing enzyme, Spastin. Several studies have indicated an interaction between reticulons and spastin, however, spastin has recently been shown to mediate contacts between the ER and lysosomes (Allison, 2017). Future studies that investigate the biochemical interaction between the reticulon family members and the role that mitotic regulatory factors play in complex formation should provide insight into mitotic ER dynamics (Diaz, 2019).

Much of the research in cell biology focused on analysis of fixed or live images to explore and understand cellular function. However, this analysis has largely relied on the individual selecting the region of analysis, thereby giving a qualitative overview of any given phenotype. Furthermore, this type of qualitative analysis, while useful, is difficult to compare with predictions from in silico modeling. Moreover, high content screening efforts require numerical measures in order to allow statistical analysis of results and identification of hits. While automated high-content image analysis has been extensively employed in studies of mammalian cells in culture, quantitative and automated methods remain under-utilized in studies of the Drosophila embryo. In order to address a quantitative approach with respect to ER movement along the perispindle region and spindle poles during mitosis, a MATLAB code was developed that allowed for the unbiased selection of ROIs at spindle poles in the syncytial embryo, and this was applied to high-content data collected with Rtnl1-GFP and ReepB-GFP embryos injected with different small molecule inhibitors. This quantitative approach gave similar results that were in line with the qualitative injection analysis, with some minor exceptions. Seemingly contrary to the qualitative observation of embryos injected with the cytoplasmic dynein inhibitor, Ciliobrevin D, the quantitative analysis carried out in this study reveals less Rtnl1-GFP enrichment to spindles and more Rtnl1-GFP depletion from the cytoplasm. An observation for this discrepancy between observations and measurements is that Rtnl1-GFP is enriched around the nuclear envelope but fails to move toward spindles upon NEB and as mitosis progresses. Similarly, the quantitative approach showed a decrease in ReepB-GFP enrichment to spindles with an increase in cytoplasmic depletion in Ciliobrevin D treatment. Furthermore, the use of a qualitative and quantitative analysis demonstrates the strength of using a mixed method approach which it is believed will greatly advance the field by providing key insights to the mechanistic underpinnings of complex cellular processes (Diaz, 2019).

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).

Reticulon-like-1, the Drosophila orthologue of the hereditary spastic paraplegia gene reticulon 2, is required for organization of endoplasmic reticulum and of distal motor axons

Several causative genes for hereditary spastic paraplegia encode proteins with intramembrane hairpin loops that contribute to the curvature of the endoplasmic reticulum (ER), but the relevance of this function to axonal degeneration is not understood. One of these genes is reticulon2. In contrast to mammals, Drosophila has only one widely expressed reticulon orthologue, Rtnl1, and therefore Drosophila was used to test its importance for ER organization and axonal function. Rtnl1 distribution overlapped with that of the ER, but in contrast to the rough ER, was enriched in axons. The loss of Rtnl1 led to the expansion of the rough or sheet ER in larval epidermis and elevated levels of ER stress. It also caused abnormalities specifically within distal portions of longer motor axons and in their presynaptic terminals, including disruption of the smooth ER (SER), the microtubule cytoskeleton and mitochondria. In contrast, proximal axon portions appeared unaffected. These results provide direct evidence for reticulon function in the organization of the SER in distal longer axons, and support a model in which spastic paraplegia can be caused by impairment of axonal the SER. These data provide a route to further understanding of both the role of the SER in axons and the pathological consequences of the impairment of this compartment (O'Sullivan, 2012).

Modeling of axonal endoplasmic reticulum network by spastic paraplegia proteins

Axons contain a smooth tubular endoplasmic reticulum (ER) network that is thought to be continuous with ER throughout the neuron; the mechanisms that form this axonal network are unknown. Mutations affecting reticulon or REEP (Drosophila Reep1) proteins, with intramembrane hairpin domains that model ER membranes, cause an axon degenerative disease, hereditary spastic paraplegia (HSP). This study shows that Drosophila axons have a dynamic axonal ER network, which these proteins help to model. Loss of HSP hairpin proteins causes ER sheet expansion, partial loss of ER from distal motor axons, and occasional discontinuities in axonal ER. Ultrastructural analysis reveals an extensive ER network in axons, which shows larger and fewer tubules in larvae that lack reticulon and REEP proteins, consistent with loss of membrane curvature. Therefore HSP hairpin-containing proteins are required for shaping and continuity of axonal ER, thus suggesting roles for ER modeling in axon maintenance and function (Yalcin, 2017).

Rtnl1 is enriched in a specialized germline ER that associates with ribonucleoprotein granule components

During oogenesis in Drosophila an organelle called the fusome plays a crucial role in germline cyst development and oocyte selection. The fusome consists of cytoskeletal proteins and intracellular membranes and, whereas many cytoskeletal components have been characterized, the nature and function of the membrane component is poorly understood. This study has found the reticulon-like 1 (Rtnl1) protein, a membrane protein resident in the endoplasmic reticulum (ER), to be highly enriched in the fusome. In other Drosophila tissues Rtnl1 marks a subset of ER membranes often derived from smooth ER. During oogenesis, Rtnl1-containing membranes are recruited to the fusome by the cytoskeletal components and become concentrated into the forming oocyte. On the central part of the fusome, which is contained within the future oocyte and also at later stages in the growing oocyte and the nurse cells, Rtnl1-containing membranes colocalize with components of ribonucleoprotein complexes that store translationally repressed mRNAs. As the ER is actively transported into the oocyte, this colocalization suggests a role for the Rtnl1-containing subdomain in anchoring the ribonucleoprotein complexes within and/or transporting them into the oocyte (Roper, 2007).

The Drosophila reticulon, Rtnl-1, has multiple differentially expressed isoforms that are associated with a sub-compartment of the endoplasmic reticulum

The reticulons are a recently discovered family of proteins that have a predominant localisation to the membrane of the endoplasmic reticulum. The precise function of the reticulons is unclear despite their presence in a wide variety of eukaryotic organisms. This study describes the characterisation of the Drosophila reticulon, reticulon-like1 (Rtnl1), which is the only functional reticulon in Drosophila. The Rtnl1 locus produces seven predicted mRNA transcripts encoding five different protein isoforms. The different transcripts have tissue-specific expression patterns remarkably similar to their mammalian counterparts. Rtnl1 protein is associated with organelles of the secretory pathway including the endoplasmic reticulum and the Golgi apparatus. Rtnl1 function appears to be non-essential or redundant since loss of function Rtnl1 mutants are viable. However, a significant reduction in life expectancy was seen in Rtnl1 mutant flies. This may point towards a possible protective role for reticulons against conditions of environmental stress (Wakefield, 2006).


Functions of Reticulon orthologs in other species

Reticulon protects the integrity of the ER membrane during ER escape of large macromolecular protein complexes

Escape of large macromolecular complexes from the endoplasmic reticulum (ER), such as a viral particle or cellular aggregate, likely induces mechanical stress initiated on the luminal side of the ER membrane, which may threaten its integrity. How the ER responds to this threat remains unknown. This study demonstrates that the cytosolic leaflet ER morphogenic protein reticulon (RTN) protects ER membrane integrity when polyomavirus SV40 escapes the ER to reach the cytosol en route to infection. SV40 coopts an intrinsic RTN function, as it was also found that RTN prevents membrane damage during ER escape of a misfolded proinsulin aggregate destined for lysosomal degradation via ER-phagy. These studies reveal that although ER membrane integrity may be threatened during ER escape of large macromolecular protein complexes, the action of RTN counters this, presumably by deploying its curvature-inducing activity to provide membrane flexibility and stability to limit mechanical stress imposed on the ER membrane (Chen, 2020).

Reticulon and CLIMP-63 regulate nanodomain organization of peripheral ER tubules

The endoplasmic reticulum (ER) is an expansive, membrane-enclosed organelle composed of smooth peripheral tubules and rough, ribosome-studded central ER sheets whose morphology is determined, in part, by the ER-shaping proteins, reticulon (RTN) and cytoskeleton-linking membrane protein 63 (CLIMP-63), respectively. Here, stimulated emission depletion (STED) super-resolution microscopy shows that reticulon4a (RTN4a) and CLIMP-63 also regulate the organization and dynamics of peripheral ER tubule nanodomains. STED imaging shows that lumenal ER monomeric oxidizing environment-optimized green fluorescent protein (ERmoxGFP), membrane Sec61betaGFP, knock-in calreticulin-GFP, and antibody-labeled ER-resident proteins calnexin and derlin-1 are all localized to periodic puncta along the length of peripheral ER tubules that are not readily observable by diffraction limited confocal microscopy. RTN4a segregates away from and restricts lumenal blob length, while CLIMP-63 associates with and increases lumenal blob length. RTN4a and CLIMP-63 also regulate the nanodomain distribution of ER-resident proteins, being required for the preferential segregation of calnexin and derlin-1 puncta away from lumenal ERmoxGFP blobs. High-speed (40 ms/frame) live cell STED imaging shows that RTN4a and CLIMP-63 regulate dynamic nanoscale lumenal compartmentalization along peripheral ER tubules. RTN4a enhances and CLIMP-63 disrupts the local accumulation of lumenal ERmoxGFP at spatially defined sites along ER tubules. The ER-shaping proteins RTN and CLIMP-63 therefore regulate lumenal ER nanodomain heterogeneity, interaction with ER-resident proteins, and dynamics in peripheral ER tubules (Gao, 2019).

Cells deploy a two-pronged strategy to rectify misfolded proinsulin aggregates

Insulin gene coding sequence mutations are known to cause mutant INS-gene-induced diabetes of youth (MIDY), yet the cellular pathways needed to prevent misfolded proinsulin accumulation remain incompletely understood. This study reports that Akita mutant proinsulin forms detergent-insoluble aggregates that entrap wild-type (WT) proinsulin in the endoplasmic reticulum (ER), thereby blocking insulin production. Two distinct quality-control mechanisms operate together to combat this insult: the ER luminal chaperone Grp170 prevents proinsulin aggregation, while the ER membrane morphogenic protein reticulon-3 (RTN3) disposes of aggregates via ER-coupled autophagy (ER-phagy). Enhanced RTN-dependent clearance of aggregated Akita proinsulin helps to restore ER export of WT proinsulin, which can promote WT insulin production, potentially alleviating MIDY. It was also found that RTN3 participates in the clearance of other mutant prohormone aggregates. Together, these results identify a series of substrates of RTN3-mediated ER-phagy, highlighting RTN3 in the disposal of pathogenic prohormone aggregates (Cunningham, 2019).

The peroxisome biogenesis factors posttranslationally target reticulon homology domain-containing proteins to the endoplasmic reticulum membrane

The endoplasmic reticulum (ER) is shaped by a class of membrane proteins containing reticulon homology domain (RHD), the conserved hydrophobic domain encompassing two short hairpin transmembrane domains. RHD resides in the outer leaflet of the ER membrane, generating high-curvature ER membrane. While most of the membrane proteins destined to enter the secretory pathway are cotranslationally targeted and inserted into ER membrane, the molecular mechanism how the RHD-containing proteins are targeted and inserted into the ER membrane remains to be clarified. This study shows that RHD-containing proteins can be posttranslationally targeted to the ER membrane. PEX19, a cytosolic peroxin, selectively recognizes the nascent RHD-containing proteins and mediates their posttranslational targeting in cooperation with PEX3, a membrane peroxin. Thus, these peroxisome biogenesis factors provide an alternative posttranslational route for membrane insertion of the RHD-containing proteins, implying that ER membrane shaping and peroxisome biogenesis may be coordinated by the posttranslational membrane insertion (Yamamoto, 2018).


REFERENCES

Search PubMed for articles about Drosophila Reticulon

Allison, R., Edgar, J. R., Pearson, G., Rizo, T., Newton, T., Gunther, S., Berner, F., Hague, J., Connell, J. W., Winkler, J., Lippincott-Schwartz, J., Beetz, C., Winner, B. and Reid, E. (2017). Defects in ER-endosome contacts impact lysosome function in hereditary spastic paraplegia. J Cell Biol 216(5): 1337-1355. PubMed ID: 28389476

Bergman, Z. J., McLaurin, J. D., Eritano, A. S., Johnson, B. M., Sims, A. Q. and Riggs, B. (2015). Spatial reorganization of the endoplasmic reticulum during mitosis relies on mitotic kinase cyclin A in the early Drosophila embryo. PLoS One 10(2): e0117859. PubMed ID: 25689737

Bobinnec, Y., Marcaillou, C., Morin, X. and Debec, A. (2003). Dynamics of the endoplasmic reticulum during early development of Drosophila melanogaster. Cell Motil Cytoskeleton 54(3): 217-225. PubMed ID: 12589680

Chen, Y. J., Williams, J. M., Arvan, P. and Tsai, B. (2020). Reticulon protects the integrity of the ER membrane during ER escape of large macromolecular protein complexes. J Cell Biol 219(2). PubMed ID: 31895406

Cunningham, C. N., Williams, J. M., Knupp, J., Arunagiri, A., Arvan, P. and Tsai, B. (2019). Cells deploy a two-pronged strategy to rectify misfolded proinsulin aggregates. Mol Cell 75(3): 442-456 e444. PubMed ID: 31176671

Diaz, U., Bergman, Z. J., Johnson, B. M., Edington, A. R., de Cruz, M. A., Marshall, W. F. and Riggs, B. (2019). Microtubules are necessary for proper Reticulon localization during mitosis. PLoS One 14(12): e0226327. PubMed ID: 31877164

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

Gao, G., Zhu, C., Liu, E. and Nabi, I. R. (2019). Reticulon and CLIMP-63 regulate nanodomain organization of peripheral ER tubules. PLoS Biol 17(8): e3000355. PubMed ID: 31469817

Gurel, P. S., Hatch, A. L. and Higgs, H. N. (2014). Connecting the cytoskeleton to the endoplasmic reticulum and Golgi. Curr Biol 24(14): R660-R672. PubMed ID: 25050967

O'Sullivan, N. C., Jahn, T. R., Reid, E. and O'Kane, C. J. (2012). Reticulon-like-1, the Drosophila orthologue of the hereditary spastic paraplegia gene reticulon 2, is required for organization of endoplasmic reticulum and of distal motor axons. Hum Mol Genet 21(15): 3356-3365. PubMed ID: 22543973

Pendin, D., McNew, J. A. and Daga, A. (2011). Balancing ER dynamics: shaping, bending, severing, and mending membranes. Curr Opin Cell Biol 23(4): 435-442. PubMed ID: 21641197

Roper, K. (2007). Rtnl1 is enriched in a specialized germline ER that associates with ribonucleoprotein granule components. J Cell Sci 120(Pt 6): 1081-1092. PubMed ID: 17327273

Schlaitz, A. L., Thompson, J., Wong, C. C., Yates, J. R., 3rd and Heald, R. (2013). REEP3/4 ensure endoplasmic reticulum clearance from metaphase chromatin and proper nuclear envelope architecture. Dev Cell 26(3): 315-323. PubMed ID: 23911198

Shibata, Y., Voss, C., Rist, J. M., Hu, J., Rapoport, T. A., Prinz, W. A. and Voeltz, G. K. (2008). The reticulon and DP1/Yop1p proteins form immobile oligomers in the tubular endoplasmic reticulum. J Biol Chem 283(27): 18892-18904. PubMed ID: 18442980

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

Wakefield, S. and Tear, G. (2006). The Drosophila reticulon, Rtnl-1, has multiple differentially expressed isoforms that are associated with a sub-compartment of the endoplasmic reticulum. Cell Mol Life Sci 63(17): 2027-2038. PubMed ID: 16847576

Yalcin, B., Zhao, L., Stofanko, M., O'Sullivan, N. C., Kang, Z. H., Roost, A., Thomas, M. R., Zaessinger, S., Blard, O., Patto, A. L., Sohail, A., Baena, V., Terasaki, M. and O'Kane, C. J. (2017).Modeling of axonal endoplasmic reticulum network by spastic paraplegia proteins. Elife 6. PubMed ID: 28742022

Yamamoto, Y. and Sakisaka, T. (2018). The peroxisome biogenesis factors posttranslationally target reticulon homology domain-containing proteins to the endoplasmic reticulum membrane. Sci Rep 8(1): 2322. PubMed ID: 29396426


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date revised: 15 March 2020

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