Small cargoes pass through synthetically glued Golgi stacks.

How are proteins transported across the stacked cisternae of the Golgi apparatus? Do they stay within the cisterna while the latter matures and progresses in an anterograde manner, or do they navigate between the cisternae via vesicles? Using synthetic biology, we engineered new tools designed to stabilize intercisternal adhesion such that Golgi cisternae are literally glued together, thus preventing any possible cisternal progression. Using bulk secretory assays and single-cell live imaging, we observed that small cargoes (but not large aggregated cargoes including collagen) still transited through glued Golgi, although the rate of transport was moderately reduced. ARF1, whose membrane recruitment is required for budding COPI vesicles, continues to cycle on and off glued Golgi. Numerous COPI-size vesicles were intercalated among the glued Golgi cisternae. These results suggest that cisternal progression is not required for anterograde transport, but do not address the possibility of cisternal maturation in situ

The multiple functions of miR-574-5p in the neuroblastoma tumor microenvironment

Neuroblastoma is the most common extracranial solid tumor in childhood and arises from neural crest cells of the developing sympathetic nervous system. Prostaglandin E2 (PGE2) has been identified as a key pro-inflammatory mediator of the tumor microenvironment (TME) that promotes neuroblastoma progression. We report that the interaction between the microRNA miR-574-5p and CUG-binding protein 1 (CUGBP1) induces the expression of microsomal prostaglandin E2 synthase 1 (mPGES-1) in neuroblastoma cells, which contributes to PGE2 biosynthesis. PGE2 in turn specifically induces the sorting of miR-574-5p into small extracellular vesicles (sEV) in neuroblastoma cell lines. sEV are one of the major players in intercellular communication in the TME. We found that sEV-derived miR-574-5p has a paracrine function in neuroblastoma. It acts as a direct Toll-like receptor 7/8 (TLR7/8) ligand and induces α-smooth muscle actin (α-SMA) expression in fibroblasts, contributing to fibroblast differentiation. This is particularly noteworthy as it has an opposite function to that in the TME of lung carcinoma, another PGE2 dependent tumor type. Here, sEV-derived miR-574-5p has an autokrine function that inhibits PGE2 biosynthesis in lung cancer cells. We report that the tetraspanin composition on the surface of sEV is associated with the function of sEV-derived miR-574-5p. This suggests that the vesicles do not only transport miRs, but also appear to influence their mode of action

Implication des sphingolipides dans le contrôle de l' autophagie (rôle de la Sphingosine 1-phosphate)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Les anomalies congénitales de glycosylation des N-glycosylprotéines

La N-glycosylation est un mécanisme post-traductionnel ubiquitaire dans les cellules de mammifères dont les grandes étapes sont maintenant bien caractérisées. La découverte d’un groupe d’erreurs innées humaines affectant ce processus a fait évoluer les idées concernant la synthèse des N-glycosylprotéines (N-GP) et a permis une meilleure compréhension de ce mécanisme complexe. Des mutations de plusieurs gènes impliqués dans la biosynthèse et la maturation des N-GP ont été caractérisées. En revanche, les liens entre les anomalies de glycosylation des N-GP et leurs répercussions cliniques n’ont pratiquement pas été explorés. Ce travail reste donc à accomplir et représente un défi qui pourrait, dans un certain nombre de cas, permettre d’envisager des voies de recherche thérapeutique.Protein N-glycosylation is a widely occurring and vital posttranslational modification in mammalian cells. Although the molecular machinery that is involved in the biosynthesis of these glycoconjugates has been largely identified, the recent discovery of a family of rare inborn diseases in which glycoproteins are abnormally glycosylated has both changed some of our ideas concerning glycoprotein biosynthesis, and given us new insights into this complex process. Advances in the diagnosis of the congenital disorders of glycosylation are well under way and mutations in several of the genes involved in the biosynthesis and maturation of N-linked glycans have been shown to underlie these diseases. By contrast, the chain of events that lead from faulty protein glycosylation to the often severe clinical presentation is an as yet unexplored aspect of these metabolic disorders, and represents a challenge for the future

Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication

International audienceThe ability of exosomes to transfer cargo from donor to acceptor cells, thereby triggering phenotypic changes in the latter, has generated substantial interest in the scientific community. However, the extent to which exosomes differ from other extracellular vesicles in terms of their biogenesis and functions remains ill-defined. Here, we discuss the current knowledge on the specificities of exosomes and other types of extracellular vesicles, and their roles as important agents of cell-to-cell communication

Quantitative characterization of extracellular vesicle uptake and content delivery within mammalian cells

International audienceExtracellular vesicles (EVs), including exosomes, are thought to mediate intercellular communication through the transfer of cargoes from donor to acceptor cells. Occurrence of EV-content delivery within acceptor cells has not been unambiguously demonstrated, let alone quantified, and remains debated. Here, we developed a cell-based assay in which EVs containing luciferase- or fluorescent-protein tagged cytosolic cargoes are loaded on unlabeled acceptor cells. Results from dose-responses, kinetics, and temperature-block experiments suggest that EV uptake is a low yield process (~1% spontaneous rate at 1 h). Further characterization of this limited EV uptake, through fractionation of membranes and cytosol, revealed cytosolic release (~30% of the uptaken EVs) in acceptor cells. This release is inhibited by bafilomycin A1 and overexpression of IFITM proteins, which prevent virus entry and fusion. Our results show that EV content release requires endosomal acidification and suggest the involvement of membrane fusion

Regulation of autophagy by sphingosine kinase 1 and its role in cell survival during nutrient starvation.

The sphingolipid ceramide induces macroautophagy (here called autophagy) and cell death with autophagic features in cancer cells. Here we show that overexpression of sphingosine kinase 1 (SK1), an enzyme responsible for the production of sphingosine 1-phosphate (S1P), in MCF-7 cells stimulates autophagy by increasing the formation of LC3-positive autophagosomes and the rate of proteolysis sensitive to the autophagy inhibitor 3-methyladenine. Autophagy was blocked in the presence of dimethylsphingosine, an inhibitor of SK activity, and in cells expressing a catalytically inactive form of SK1. In SK1(wt)-overexpressing cells, however, autophagy was not sensitive to fumonisin B1, an inhibitor of ceramide synthase. In contrast to ceramide-induced autophagy, SK1(S1P)-induced autophagy is characterized by (i) the inhibition of mammalian target of rapamycin signaling independently of the Akt/protein kinase B signaling arm and (ii) the lack of robust accumulation of the autophagy protein Beclin 1. In addition, nutrient starvation induced both the stimulation of autophagy and SK activity. Knocking down the expression of the autophagy protein Atg7 or that of SK1 by siRNA abolished starvation-induced autophagy and increased cell death with apoptotic hallmarks. In conclusion, these results show that SK1(S1P)-induced autophagy protects cells from death with apoptotic features during nutrient starvation

Land-locked mammalian Golgi reveals cargo transport between stable cisternae

The different composition of Golgi cisternae gave rise to two different models for intra-Golgi traffic: one where stable cisternae communicate via vesicles and another one where cisternae biochemically mature to ensure anterograde transport. Here, the authors provide evidence in support of the stable compartments model

Extracellular vesicle-based therapeutics : natural versus engineered targeting and trafficking

Extracellular vesicles (EVs) are increasingly being recognized as mediators of intercellular signaling via the delivery of effector molecules. Interestingly, certain types of EVs are also capable of inducing therapeutic responses. For these reasons, the therapeutic potential of EVs is a topic of intense research, both in the context of drug delivery and regenerative medicine. However, to fully utilize EVs for therapeutic purposes, an improved understanding of the mechanisms by which they function would be highly advantageous. Here, the current state of knowledge regarding the cellular uptake and trafficking of EVs is reviewed, along with a consideration of how these pathways potentially influence the functions of therapeutic EVs. Furthermore, the natural cell-targeting abilities, biodistribution profiles, and pharmacokinetics of exogenously administered EVs, along with the components responsible for these features are discussed. An overview of the potential clinical applications and preclinical examples of their successful use is also provided. Finally, examples of EV modifications that have successfully been employed to improve their therapeutic characteristics receive a particular focus. We suggest that, in addition to investigation of EV cell targeting and routes of uptake, future research into the routes of intracellular trafficking in recipient cells is required to optimally utilize EVs for therapeutic purposes

The multiple functions of miR-574-5p in the neuroblastoma tumor microenvironment

Neuroblastoma is the most common extracranial solid tumor in childhood and arises from neural crest cells of the developing sympathetic nervous system. Prostaglandin E₂ (PGE₂) has been identified as a key pro-inflammatory mediator of the tumor microenvironment (TME) that promotes neuroblastoma progression. We report that the interaction between the microRNA miR-574-5p and CUG-binding protein 1 (CUGBP1) induces the expression of microsomal prostaglandin E₂ synthase 1 (mPGES-1) in neuroblastoma cells, which contributes to PGE₂ biosynthesis. PGE₂ in turn specifically induces the sorting of miR-574-5p into small extracellular vesicles (sEV) in neuroblastoma cell lines. sEV are one of the major players in intercellular communication in the TME. We found that sEV-derived miR-574-5p has a paracrine function in neuroblastoma. It acts as a direct Toll-like receptor 7/8 (TLR7/8) ligand and induces α-smooth muscle actin (α-SMA) expression in fibroblasts, contributing to fibroblast differentiation. This is particularly noteworthy as it has an opposite function to that in the TME of lung carcinoma, another PGE₂ dependent tumor type. Here, sEV-derived miR-574-5p has an autokrine function that inhibits PGE₂ biosynthesis in lung cancer cells. We report that the tetraspanin composition on the surface of sEV is associated with the function of sEV-derived miR-574-5p. This suggests that the vesicles do not only transport miRs, but also appear to influence their mode of action