Identification and characterization of atlastin interacting partners involved in maintaining and determining the morphology of endoplasmic reticulum in Drosophila melanogaster

The endoplasmic reticulum (ER) is a complex membrane network that undergoes continuous remodeling while retaining its overall structure. Drosophila atlastin localizes specifically to the ER and it has been demonstrated to be the GTPase responsible for the homotypic fusion of ER membranes. Recently it has been shown that atlastin interacts with other ER tubule-forming proteins such as reticulon and DP1/REEP/Yop1 families. These families show little overall sequence homology but they share a conserved domain of about 200 amino acids (Reticulon Homology Domain, RHD) that includes two hydrophobic segments that seems to form a hairpin in the membrane. The hydrophobic portions of these ER-shaping proteins appear to occupy the outer leaflet of the phospholipid bilayer, possibly generating curvature via hydrophobic wedging. Using Drosophila melanogaster we studied the function of reticulon (Rtnl1) and DP1 in maintaining and determining the morphology of the ER. We found that in Drosophila Rtnl1 and atlastin interact genetically in an antagonistic manner and that modulation of Rtnl1 expression in vivo markedly affects atlastin loss and gain of function phenotypes. Indeed, we demonstrated that in Drosophila genetic elimination of Rtnl1 in the atlastin null background rescues the lethality associated with depletion of atlastin. This genetic interaction between Rtnl1 and atlastin is also supported by experiments in the Drosophila eye: ectopic expression of atlastin in the eye causes a small eye phenotype and RNAi mediated loss of Rtnl1 in an eye expressing atlastin results in enhancement of the atlastin dependent small eye phenotype. This antagonistic genetic interaction between Rtnl1 and atlastin suggests that these two proteins exert opposing functions in the control of ER architecture. Consistent with this hypothesis we found that loss of Rtnl1 leads to elongation of ER profiles while its overexpression produces shorter profiles. Moreover, FLIP experiments suggest that the ER lumen is discontinuous in Drosophila tissues overexpressing Rtnl1, further corroborating the hypothesis that Rtnl1 functions to counterbalance atlastin fusogenic activity by facilitating membrane fission to maintain the morphology of the ER. This activity was confirmed in vitro by showing that Rtnl1 reconstituted into giant unilamellar vesicles is sufficient to trigger membrane budding and production of vesicles. Our studies of DP1 in Drosophila demonstrated that an antagonistic genetic interaction exists also between DP1 and atlastin. Indeed, such interaction is obvious both in the fly eye and in cell culture. Overexpression of DP1 in an eye simultaneously expressing atlastin resulted in a rescue of the atlastin-dependent phenotype and the hyperfusion phenotype caused by atlastin overexpression in COS-7 cells is rescued by coexpressing DP1. Moreover, we found that in Drosophila DP1 influences the morphology of the ER since neurons lacking DP1 display an elongation of the ER profiles. Thus, DP1 seems to have a function analogous to that of Rtnl1. This observation suggests that the membrane fusion mediated by atlastin is counterbalanced by the activity of two or possibly more proteins in order to maintain the general morphology of the ER network. Since it has been demonstrated that the RHD is the crucial region of reticulon and DP1, we propose that proteins containing this domain, such as reticulon and DP1/REEP/Yop1 proteins, could have an intrinsic ability to break ER membranes due to their capacity to induce extreme curvature of the lipid bilayers. Regions of extreme curvature can potentially be the sites of membrane scission because of the intrinsic instability of lipids. Our work suggests that a balance between membrane fusion and scission events is required to maintain the overall structure of the ER network and identifies potential candidate proteins with fission promoting activity

Dynamic constriction andfission of endoplasmicreticulum 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. Here we find 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.This work was partially supported by NIH R01GM121725 to V.A.F., a 5x1000 grant from the Italian Ministry of Health and Telethon GGP11189 to A.D., Spanish Ministry of Science, Innovation and Universities grants BFU2015-70552-P to V.A.F. and A.V.S., and BFU2015-63714-R and PGC2018-099341-B-I00 to B.I., Basque Government grant IT1196-19, Russian Science Foundation Grant No. 17-75-30064 and Ministry of Science and Higher Education of the Russian Federation

Identification and characterization of atlastin interacting partners involved in maintaining and determining the morphology of endoplasmic reticulum in Drosophila melanogaster

The endoplasmic reticulum (ER) is a complex membrane network that undergoes continuous remodeling while retaining its overall structure. Drosophila atlastin localizes specifically to the ER and it has been demonstrated to be the GTPase responsible for the homotypic fusion of ER membranes. Recently it has been shown that atlastin interacts with other ER tubule-forming proteins such as reticulon and DP1/REEP/Yop1 families. These families show little overall sequence homology but they share a conserved domain of about 200 amino acids (Reticulon Homology Domain, RHD) that includes two hydrophobic segments that seems to form a hairpin in the membrane. The hydrophobic portions of these ER-shaping proteins appear to occupy the outer leaflet of the phospholipid bilayer, possibly generating curvature via hydrophobic wedging. Using Drosophila melanogaster we studied the function of reticulon (Rtnl1) and DP1 in maintaining and determining the morphology of the ER. We found that in Drosophila Rtnl1 and atlastin interact genetically in an antagonistic manner and that modulation of Rtnl1 expression in vivo markedly affects atlastin loss and gain of function phenotypes. Indeed, we demonstrated that in Drosophila genetic elimination of Rtnl1 in the atlastin null background rescues the lethality associated with depletion of atlastin. This genetic interaction between Rtnl1 and atlastin is also supported by experiments in the Drosophila eye: ectopic expression of atlastin in the eye causes a small eye phenotype and RNAi mediated loss of Rtnl1 in an eye expressing atlastin results in enhancement of the atlastin dependent small eye phenotype. This antagonistic genetic interaction between Rtnl1 and atlastin suggests that these two proteins exert opposing functions in the control of ER architecture. Consistent with this hypothesis we found that loss of Rtnl1 leads to elongation of ER profiles while its overexpression produces shorter profiles. Moreover, FLIP experiments suggest that the ER lumen is discontinuous in Drosophila tissues overexpressing Rtnl1, further corroborating the hypothesis that Rtnl1 functions to counterbalance atlastin fusogenic activity by facilitating membrane fission to maintain the morphology of the ER. This activity was confirmed in vitro by showing that Rtnl1 reconstituted into giant unilamellar vesicles is sufficient to trigger membrane budding and production of vesicles. Our studies of DP1 in Drosophila demonstrated that an antagonistic genetic interaction exists also between DP1 and atlastin. Indeed, such interaction is obvious both in the fly eye and in cell culture. Overexpression of DP1 in an eye simultaneously expressing atlastin resulted in a rescue of the atlastin-dependent phenotype and the hyperfusion phenotype caused by atlastin overexpression in COS-7 cells is rescued by coexpressing DP1. Moreover, we found that in Drosophila DP1 influences the morphology of the ER since neurons lacking DP1 display an elongation of the ER profiles. Thus, DP1 seems to have a function analogous to that of Rtnl1. This observation suggests that the membrane fusion mediated by atlastin is counterbalanced by the activity of two or possibly more proteins in order to maintain the general morphology of the ER network. Since it has been demonstrated that the RHD is the crucial region of reticulon and DP1, we propose that proteins containing this domain, such as reticulon and DP1/REEP/Yop1 proteins, could have an intrinsic ability to break ER membranes due to their capacity to induce extreme curvature of the lipid bilayers. Regions of extreme curvature can potentially be the sites of membrane scission because of the intrinsic instability of lipids. Our work suggests that a balance between membrane fusion and scission events is required to maintain the overall structure of the ER network and identifies potential candidate proteins with fission promoting activity.Il reticolo endoplasmatico (ER) è un organello altamente dinamico formato da un complesso sistema di membrane in continuo movimento e rimodellamento. La biogenesi ed il mantenimento dell’elaborata architettura dell’ER sono fondamentali per il corretto svolgimento delle sue funzioni e dipendono da eventi di fusione e di fissione delle membrane e dall’azione di proteine capaci di rimodellare le membrane. La fusione omotipica delle membrane dell’ER dipende dalla proteina atlastina, una GTPasi localizzata nelle membrane dell’ER. Al contrario, i meccanismi e le proteine coinvolte nella fissione delle membrane sono ancora sconosciuti. Recentemente, è stato dimostrato che atlastina interagisce con proteine appartenenti alle famiglie reticulons e DP1/REEP/Yop1, proteine coinvolte nel determinare la morfologia dell’ER. Queste proteine, sebbene appartenenti a famiglie differenti, posseggono un dominio altamente conservato di circa 200 aminoacidi (chiamato RHD) costituito da due domini transmembrana separati da una breve ansa citosolica. È stato proposto che le due porzioni idrofobiche si inseriscano nel foglietto esterno del doppio strato fosfolipidico in una struttura a forcina; tale struttura causerebbe quindi una deformazione del monostrato esterno della membrana, generando una curvatura localizzata della membrana. In questa tesi, utilizzando come organismo modello Drosophila melanogaster, abbiamo studiato il ruolo delle proteine reticulon-1 (Rtnl1) e DP1 nel generare e mantenere la complessa architettura dell’ER. Esperimenti in vivo hanno dimostrato che in Drosophila esiste una forte interazione genetica antagonistica tra Rtnl1 e atlastina. Infatti, i nostri risultati dimostrano che la letalità causata dall’assenza del gene atlastina è recuperata dalla simultanea perdita di funzione di Rtnl1. Questa interazione tra Rtnl1 e atlastina è stata confermata anche da esperimenti condotti nell’occhio di Drosophila: un’espressione ectopica di atlastina nell’occhio di Drosophila causa un occhio piccolo e rovinato; l’assenza di Rtnl1 in un occhio che contemporaneamente sovraesprime atlastina porta ad un peggioramento del fenotipo dell’occhio che diventa ancor più rovinato. Questa forte interazione genetica tra Rtnl1 e atlastina suggerisce che queste due proteine abbiano funzioni opposte nel mantenimento dell’architettura dell’ER. Inoltre, abbiamo dimostrato che l’assenza di Rtnl1 in vivo provoca l’allungamento dei profili dell’ER mentre, al contrario, la sua sovraespressione causa frammentazione e perdita della normale continuità del lume dell’ER. Questi risultati avvalorano ulteriormente l’ipotesi che Rtnl1 sia in grado di controbilanciare l’attività di fusione mediata da atlastina probabilmente facilitando il processo di fissione delle membrane dell’ER. Questa ipotesi è stata confermata da esperimenti condotti in vitro: Rtnl1, infatti, è in grado di promuovere autonomamente il “budding” di membrana e la produzione di vescicole. Abbiamo dimostrato che esiste una interazione genetica antagonistica anche tra DP1 e atlastina in Drosophila. Infatti, la sovraespressione simultanea di DP1 e atlastina nell'occhio porta ad un recupero del fenotipo “occhio rovinato” causato dall'espressione di atlastina. Inoltre, il fenotipo di iperfusione dell’ER causato dalla sovraespressione di atlastina in cellule COS-7 viene recuperato co-esprimendo DP1. Abbiamo anche dimostrato che DP1 è coinvolto nel mantenimento della morfologia dell’ER dato che neuroni privi di DP1 presentano profili dell’ER mediamente più lunghi rispetto a neuroni di controllo. DP1, quindi, sembra avere una funzione simile a quella di Rtnl1. Questi risultati suggeriscono che la fusione delle membrane dell’ER mediata da atlastina sembra essere controbilanciata dall’attività di due o più proteine che cooperano per mantenere la normale morfologia dell’ER. Dato che è stato dimostrato che il dominio RHD è la regione importante per la funzione di Rtnl1 e DP1, ipotizziamo che le proteine che contengono questo particolare dominio possano avere l’intrinseca abilità di rompere le membrane dell’ER. Questa abilità è dovuta alla capacità di queste proteine di indurre un’estrema curvatura delle membrane; a causa dell’intrinseca instabilità dei lipidi le regioni di estrema curvatura possono potenzialmente essere il punto di rottura delle membrane. I dati da noi ottenuti suggeriscono che un equilibrio tra eventi di fusione e di fissione delle membrane sia necessario per mantenere la corretta morfologia dell’ER e identificano due proteine, Rtnl1 e DP1, che sono coinvolte nel promuovere gli eventi di fissione delle membrane dell’ER

Lung Involvement in Systemic Juvenile Idiopathic Arthritis: A Narrative Review

Systemic juvenile idiopathic arthritis associated with lung disorders (sJIA-LD) is a subtype of sJIA characterized by the presence of chronic life-threatening pulmonary disorders, such as pulmonary hypertension, interstitial lung disease, pulmonary alveolar proteinosis and/or endogenous lipoid pneumonia, which were exceptionally rare before 2013. Clinically, these children show a striking dissociation between the relatively mild clinical manifestations (tachypnoea, clubbing and chronic cough) and the severity of the pulmonary inflammatory process. Our review describes sJIA-LD as having a reported prevalence of approximately 6.8%, with a mortality rate of between 37% and 68%. It is often associated with an early onset (<2 years of age), macrophage activation syndrome and high interleukin (IL)-18 circulating levels. Other risk factors may be trisomy 21 and a predisposition to adverse reactions to biological drugs. The most popular hypothesis is that the increase in the number of sJIA-LD cases can be attributed to the increased use of IL-1 and IL-6 blockers. Two possible explanations have been proposed, named the “DRESS hypothesis” and the “cytokine plasticity hypothesis”. Lung ultrasounds and the intercellular-adhesion-molecule-5 assay seem to be promising tools for the early diagnosis of sJIA-LD, although high resolution computed tomography remains the gold standard. In this review, we also summarize the treatment options for sJIA-LD, focusing on JAK inhibitors