Torsins are developmentally essential AAA+ proteins, and mutation of human torsinA causes the neurological disease DYT1 dystonia. They localize in the ER membranes, but their cellular function remains unclear. We now show that dTorsin is required in Drosophila adipose tissue, where it suppresses triglyceride levels, promotes cell growth, and elevates membrane lipid content. We also see that human torsinA at the inner nuclear membrane is associated with membrane expansion and elevated cellular lipid content. Furthermore, the key lipid metabolizing enzyme, lipin, is mislocalized in dTorsin-KO cells, and dTorsin increases levels of the lipin substrate, phosphatidate, and reduces the product, diacylglycerol. Finally, genetic suppression of dLipin rescues dTorsin-KO defects, including adipose cell size, animal growth, and survival. These findings identify that torsins are essential regulators of cellular lipid metabolism and implicate disturbed lipid biology in childhood-onset DYT1 dystonia.
Repeat expansions in the C9orf72 gene cause amyotrophic lateral sclerosis and frontotemporal dementia characterized by dipeptide-repeat protein (DPR) inclusions. The toxicity associated with two of these DPRs, poly-GR and poly-PR, has been associated with nucleocytoplasmic transport. To investigate the causal role of poly-GR or poly-PR on active nucleocytoplasmic transport, we measured nuclear import and export in poly-GR or poly-PR expressing Hela cells, neuronal-like SH-SY5Y cells and iPSC-derived motor neurons. Our data strongly indicate that poly-GR and poly-PR do not directly impede active nucleocytoplasmic transport.
Energy metabolism has been repeatedly linked to amyotrophic lateral sclerosis (ALS). Yet, motor neuron (MN) metabolism remains poorly studied and it is unknown if ALS MNs differ metabolically from healthy MNs. To address this question, we first performed a metabolic characterization of induced pluripotent stem cells (iPSCs) versus iPSC-derived MNs and subsequently compared MNs from ALS patients carrying FUS mutations to their CRISPR/Cas9-corrected counterparts. We discovered that human iPSCs undergo a lactate oxidation-fuelled prooxidative metabolic switch when they differentiate into functional MNs. Simultaneously, they rewire metabolic routes to import pyruvate into the TCA cycle in an energy substrate specific way. By comparing patient-derived MNs and their isogenic controls, we show that ALS-causing mutations in FUS did not affect glycolytic or mitochondrial energy metabolism of human MNs in vitro. These data show that metabolic dysfunction is not the underlying cause of the ALS-related phenotypes previously observed in these MNs.
The lipid metabolite diacylglycerol (DAG) is required for transport carrier biogenesis at the Golgi, although how cells regulate its levels is not well understood. Phospholipid synthesis involves highly regulated pathways that consume DAG and can contribute to its regulation. Here we altered phosphatidylcholine (PC) and phosphatidylinositol synthesis for a short period of time in CHO cells to evaluate the changes in DAG and its effects in membrane trafficking at the Golgi. We found that cellular DAG rapidly increased when PC synthesis was inhibited at the non-permissive temperature for the rate-limiting step of PC synthesis in CHO-MT58 cells. DAG also increased when choline and inositol were not supplied. The major phospholipid classes and triacylglycerol remained unaltered for both experimental approaches. The analysis of Golgi ultrastructure and membrane trafficking showed that 1) the accumulation of the budding vesicular profiles induced by propanolol was prevented by inhibition of PC synthesis, 2) the density of KDEL receptor-containing punctated structures at the endoplasmic reticulum-Golgi interface correlated with the amount of DAG, and 3) the postGolgi transport of the yellow fluorescent temperature-sensitive G protein of stomatitis virus and the secretion of a secretory form of HRP were both reduced when DAG was lowered. We confirmed that DAG-consuming reactions of lipid synthesis were present in Golgi-enriched fractions. We conclude that phospholipid synthesis pathways play a significant role to regulate the DAG required in Golgi-dependent membrane trafficking.
Background: III spectrin function at the Golgi remains unclear. Results: III spectrin is enriched in distal Golgi compartments and supports anterograde transport. PI4P is determinant for the III spectrin association with Golgi membranes. Conclusion: III spectrin is necessary for the structural and functional organization of the Golgi. Significance: We provide new in vivo insights of the role of III spectrin at the Golgi.
Diacylglycerol (DAG) is required for membrane traffic and structural organization at the Golgi. DAG is a lipid metabolite of several enzymatic reactions present at this organelle, but the mechanisms by which they are regulated are still unknown. Here, we show that cargo arrival at the Golgi increases the recruitment of the DAG-sensing constructs C1-PKCθ-GFP and the PKD-wt-GFP. The recruitment of both constructs was reduced by PLCγ1 silencing. Post-Golgi trafficking of transmembrane and soluble proteins was impaired in PLCγ1-silenced cells. Under basal conditions, PLCγ1 contributed to the maintenance of the pool of DAG associated with the Golgi and to the structural organization of the organelle. Finally, we show that cytosolic phospholipase C (PLC) can hydrolyse phosphatidylinositol 4-phosphate in isolated Golgi membranes. Our results indicate that PLCγ1 is part of the molecular mechanism that couples cargo arrival at the Golgi with DAG production to co-ordinate the formation of transport carriers for post-Golgi traffic.
We previously reported that actin-depolymerizing agents promote the alkalization of the Golgi stack and the trans-Golgi network. The main determinant of acidic pH at the Golgi is the vacuolar-type H ؉ -translocating ATPase (V-ATPase), whose V 1 domain subunits B and C bind actin. We have generated a GFPtagged subunit B2 construct (GFP-B2) that is incorporated into the V 1 domain, which in turn is coupled to the V 0 sector. GFP-B2 subunit is enriched at distal Golgi compartments in HeLa cells. Subcellular fractionation, immunoprecipitation, and inversal FRAP experiments show that the actin depolymerization promotes the dissociation of V 1 -V 0 domains, which entails subunit B2 translocation from Golgi membranes to the cytosol. Moreover, molecular interaction between subunits B2 and C1 and actin were detected. In addition, Golgi membrane lipid order disruption by D-ceramide-C6 causes Golgi pH alkalization. We conclude that actin regulates the Golgi pH homeostasis maintaining the coupling of V 1 -V 0 domains of V-ATPase through the binding of microfilaments to subunits B and C and preserving the integrity of detergent-resistant membrane organization. These results establish the Golgi-associated V-ATPase activity as the molecular link between actin and the Golgi pH.The secretory pathway is characterized by progressive lumen acidification of its organelles, from almost neutral in the endoplasmic reticulum (ER) 4 (pH Ϸ7.1-7.2), along the cis-to-trans Golgi stack (pH Ϸ6.7-6.0), to more acidic in the trans-Golgi network (TGN) and secretory vesicles/granules (pH Ϸ5.0) (1-3). This pH gradient is crucial for post-translational modifications and membrane trafficking events (4, 5). The main molecular determinant of the progressive fall in pH along the secretory pathway is the vacuolar [H ϩ ]ATPase (V-ATPase) (6 -8). V-ATPase is a multisubunit complex composed of two large domains, V 0 and V 1 . The V 0 domain is a 260-kDa integral membrane complex made up of five different subunits (a, b, c, cЈ, cЉ, d, and e), which mediates proton translocation; the V 1 domain is a 600 -650-kDa peripheral complex composed of eight different subunits (A, B, C, D, E, F, G, and H), which is responsible for the ATP hydrolysis that provides the mechanical force necessary for proton (H ϩ ) translocation (7, 9 -11). Whereas they are the primary source of proton delivery to endomembranes consuming ATP, the final steady-state pH in the secretory pathway is the result of the balance between active H ϩ pumping by the V-ATPase, passive H ϩ efflux through organelle endogenous H ϩ permeability, and differences in counter-ion conductance (3, 12).How differences in the pH of individual secretory compartments are generated is not well understood. Differential VATPase density and/or local regulatory mechanisms in secretory organelles and subcompartments are possible (13). In this respect, V-ATPase-dependent proton translocation could be regulated by several mechanisms, which include the following: (a) differential V-ATPase subunit expression; (b) intracel...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.