Patient-derived glioblastoma stem cells transfer mitochondria through tunneling nanotubes in tumor organoids.

Glioblastoma (GBM) is the most aggressive brain cancer and its relapse after surgery, chemo and radiotherapy appears to be led by GBM stem cells (GSCs). Also, tumor networking and intercellular communication play a major role in driving GBM therapy-resistance. Tunneling Nanotubes (TNTs), thin membranous open-ended channels connecting distant cells, have been observed in several types of cancer, where they emerge to drive a more malignant phenotype. Here, we investigated whether GBM cells are capable to intercommunicate by TNTs. Two GBM stem-like cells (GSLCs) were obtained from the external and infiltrative zone of one GBM from one patient. We show, for the first time, that both GSLCs, grown in classical 2D culture and in 3D-tumor organoids, formed functional TNTs which allowed mitochondria transfer. In the organoid model, recapitulative of several tumor's features, we observed the formation of a network between cells constituted of both Tumor Microtubes (TMs), previously observed in vivo, and TNTs. In addition, the two GSLCs exhibited different responses to irradiation in terms of TNT induction and mitochondria transfer, although the correlation with the disease progression and therapy-resistance needs to be further addressed. Thus, TNT-based communication is active in different GSLCs derived from the external tumoral areas associated to GBM relapse, and we propose that they participate together with TMs in tumor networking.

Fate and propagation of endogenously formed Tau aggregates in neuronal cells.

Tau accumulation in the form of neurofibrillary tangles in the brain is a hallmark of tauopathies such as Alzheimer's disease (AD). Tau aggregates accumulate in brain regions in a defined spatiotemporal pattern and may induce the aggregation of native Tau in a prion-like manner. However, the underlying mechanisms of cell-to-cell spreading of Tau pathology are unknown and could involve encapsulation within exosomes, trans-synaptic passage, and tunneling nanotubes (TNTs). We have established a neuronal cell model to monitor both internalization of externally added fibrils, synthetic (K18) or Tau from AD brain extracts, and real-time conversion of microtubule-binding domain of Tau fused to a fluorescent marker into aggregates. We found that these endogenously formed deposits colabel with ubiquitin and p62 but are not recruited to macroautophagosomes, eventually escaping clearance. Furthermore, endogenous K18-seeded Tau aggregates spread to neighboring cells where they seed new deposits. Transfer of Tau aggregates depends on direct cell contact, and they are found inside TNTs connecting neuronal cells. We further demonstrate that contact-dependent transfer occurs in primary neurons and between neurons and astrocytes in organotypic cultures.

Tunneling Nanotubes: The Fuel of Tumor Progression?

Tunneling nanotubes (TNTs) are thin membrane tubes connecting remote cells and allowing the transfer of cellular content. TNTs have been reported in several cancer in vitro, ex vivo, and in vivo models. Cancer cells exploit TNT-like connections to exchange material between themselves or with the tumoral microenvironment. Cells acquire new abilities (e.g., enhanced metabolic plasticity, migratory phenotypes, angiogenic ability, and therapy resistance) via these exchanges, contributing to cancer aggressiveness. Here, we review the morphological and functional features of TNT-like structures and their impact on cancer progression and resistance to therapies. Finally, we discuss the case of glioblastoma (GBM), in which a functional and resistant network between cancer cells in an in vivo model has been described for the first time.

TspanC8 tetraspanins differentially regulate ADAM10 endocytosis and half-life.

ADAM10 is a transmembrane metalloprotease that is essential for development and tissue homeostasis. It cleaves the ectodomain of many proteins, including amyloid precursor protein, and plays an essential role in Notch signaling. ADAM10 associates with six members of the tetraspanin superfamily referred to as TspanC8 (Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33), which regulate its exit from the endoplasmic reticulum and its substrate selectivity. We now show that ADAM10, Tspan5, and Tspan15 influence each other's expression level. Notably, ADAM10 undergoes faster endocytosis in the presence of Tspan5 than in the presence of Tspan15, and Tspan15 stabilizes ADAM10 at the cell surface yielding high expression levels. Reciprocally, ADAM10 stabilizes Tspan15 at the cell surface, indicating that it is the Tspan15/ADAM10 complex that is retained at the plasma membrane. Chimeric molecules indicate that the cytoplasmic domains of these tetraspanins contribute to their opposite action on ADAM10 trafficking and Notch signaling. In contrast, an unusual palmitoylation site at the end of Tspan15 C-terminus is dispensable. Together, these findings uncover a new level of ADAM10 regulation by TspanC8 tetraspanins.

Pancreatic Cell Fate Determination Relies on Notch Ligand Trafficking by NFIA.

Notch is activated globally in pancreatic progenitors; however, for progenitors to differentiate into endocrine cells, they must escape Notch activation to express Neurogenin-3. Here, we find that the transcription factor nuclear factor I/A (NFIA) promotes endocrine development by regulating Notch ligand Dll1 trafficking. Pancreatic deletion of NFIA leads to cell fate defects, with increased duct and decreased endocrine formation, while ectopic expression promotes endocrine formation in mice and human pancreatic progenitors. NFIA-deficient mice exhibit dysregulation of trafficking-related genes including increased expression of Mib1, which acts to target Dll1 for endocytosis. We find that NFIA binds to the Mib1 promoter, with loss of NFIA leading to an increase in Dll1 internalization and enhanced Notch activation with rescue of the cell fate defects after Mib1 knockdown. This study reveals NFIA as a pro-endocrine factor in the pancreas, acting to repress Mib1, inhibit Dll1 endocytosis and thus promote escape from Notch activation.

Ligand-activated Notch undergoes DTX4-mediated ubiquitylation and bilateral endocytosis before ADAM10 processing.

The Notch signaling pathway, which is activated by cell-cell contact, is a major regulator of cell fate decisions. Mammalian Notch1 is present at the cell surface as a heterodimer of the Notch extracellular domain associated with the transmembrane and intracellular domains. After ligand binding, Notch undergoes proteolysis, releasing the Notch intracellular domain (NICD) that regulates gene expression. We monitored the early steps of activation with biochemical analysis, immunofluorescence analysis, and live-cell imaging of Notch1-expressing cells. We found that, upon ligand binding, Notch1 at the cell surface was ubiquitylated by the E3 ubiquitin ligase DTX4. This ubiquitylation event led to the internalization of the Notch1 extracellular domain by the ligand-expressing cell and the internalization of the membrane-anchored fragment of Notch1 and DTX4 by the Notch1-expressing cell, which we referred to as bilateral endocytosis. ADAM10 generates a cleavage product of Notch that is necessary for the formation of the NICD, which has been thought to occur at the cell surface. However, we found that blocking dynamin-mediated endocytosis of Notch1 and DTX4 reduced the colocalization of Notch1 with ADAM10 and the formation of the ADAM10-generated cleavage product of Notch1, suggesting that ADAM10 functions in an intracellular compartment to process Notch. Thus, this study suggests that a specific pool of ADAM10 acts on Notch in an endocytic compartment, rather than at the cell surface.

TspanC8 tetraspanins differentially regulate the cleavage of ADAM10 substrates, Notch activation and ADAM10 membrane compartmentalization.

The metalloprotease ADAM10 mediates the shedding of the ectodomain of various cell membrane proteins, including APP, the precursor of the amyloid peptide Aß, and Notch receptors following ligand binding. ADAM10 associates with the members of an evolutionary conserved subgroup of tetraspanins, referred to as TspanC8, which regulate its exit from the endoplasmic reticulum. Here we show that 4 of these TspanC8 (Tspan5, Tspan14, Tspan15 and Tspan33) which positively regulate ADAM10 surface expression levels differentially impact ADAM10-dependent Notch activation and the cleavage of several ADAM10 substrates, including APP, N-cadherin and CD44. Sucrose gradient fractionation, single molecule tracking and quantitative mass-spectrometry analysis of the repertoire of molecules co-immunoprecipitated with Tspan5, Tspan15 and ADAM10 show that these two tetraspanins differentially regulate ADAM10 membrane compartmentalization. These data represent a unique example where several tetraspanins differentially regulate the function of a common partner protein through a distinct membrane compartmentalization.

Tracking trafficking of Notch and its ligands in mammalian cells.

The Notch receptor and its ligands are cell surface transmembrane proteins that are internalized. Endocytosis and vesicle trafficking play key roles in Notch signaling activation and modulation. In mammalian cultured cells it is possible to track these cell surface molecules by pulse-labeling these proteins in vivo. One labeling protocol consists in the covalent linkage of membrane-impermeable biotin followed by western blotting. An alternative protocol consists of using high affinity antibodies against the extracellular domains of the proteins followed by immunofluorescence, thereby allowing monitoring of the fate of the labeled proteins. In this chapter, we will describe these two approaches to study the dynamics of receptor and ligand trafficking.

Α-arrestins - new players in Notch and GPCR signaling pathways in mammals.

For many years, ß-arrestins have been known to be involved in G-protein-coupled receptor (GPCR) desensitization. However, ß-arrestins belong to a family of proteins that act as multifunctional scaffolding proteins, in particular during trafficking of transmembrane receptors. The arrestin family comprises visual arrestins, ß-arrestins and α-arrestins. In mammals, the functions of the α-arrestins are beginning to be elucidated, and they are described as versatile adaptors that link GPCRs or the Notch receptor to E3 ubiquitin ligases and endocytic factors. These α-arrestins can act in sequence, complementarily or cooperatively with ß-arrestins in trafficking and ubiquitylation events. This Commentary will summarize the recent advances in our understanding of the functions and properties of these α-arrestin proteins in comparison to ß-arrestins, and will highlight a new hypothesis linking their functional complementarity to their physical interactions. α- and ß-arrestins could form transient and versatile heterodimers that form a bridge between cargo and E3 ubiquitin ligases, thus allowing trafficking to proceed.

Α-arrestin 1 (ARRDC1) and ß-arrestins cooperate to mediate Notch degradation in mammals.

Notch signaling is a conserved signaling pathway implicated in embryogenesis and adult tissue maintenance. Notch signaling strength is strictly regulated, notably by maintaining a controlled pool of functional receptor at the cell surface. Mammalian non-activated Notch receptor is internalized, ubiquitylated by the Itch E3 ubiquitin ligase and degraded in the lysosomes. Here, we show that ß-arrestins are necessary for Itch-Notch interaction and for Itch-driven ubiquitylation and degradation of Notch. Interestingly, ß-arrestins do not directly bind Itch but heterodimerize with a member of another subfamily of arrestins called ARRDC1 or α-arrestin 1, which harbors PPxY motifs that allow direct interaction with Itch. Cells transfected with ARRDC1 mutated in PPxY motifs show reduced Itch-mediated Notch ubiquitylation and impaired lysosomal degradation of Notch, as observed in ß-arrestin(-/-) or Itch(-/-) cells. Our data show for the first time that ARRDC1 and ß-arrestins heterodimerize and cooperate in the same complex to promote non-activated Notch receptor degradation, thus acting as negative regulators of Notch signaling.

Ubiquitinations in the notch signaling pathway.

The very conserved Notch pathway is used iteratively during development and adulthood to regulate cell fates. Notch activation relies on interactions between neighboring cells, through the binding of Notch receptors to their ligands, both transmembrane molecules. This inter-cellular contact initiates a cascade of events eventually transforming the cell surface receptor into a nuclear factor acting on the transcription of specific target genes. This review highlights how the various processes undergone by Notch receptors and ligands that regulate the pathway are linked to ubiquitination events.

The ubiquitin-specific protease 12 (USP12) is a negative regulator of notch signaling acting on notch receptor trafficking toward degradation.

Notch signaling is critical for development and adult tissue physiology, controlling cell fate in a context-dependent manner. Upon ligand binding, the transmembrane Notch receptor undergoes two ordered proteolytic cleavages releasing Notch intracellular domain, which regulates the transcription of Notch target genes. The strength of Notch signaling is of crucial importance and depends notably on the quantity of Notch receptor at the cell surface. Using an shRNA library screen monitoring Notch trafficking and degradation in the absence of ligand, we identified mammalian USP12 and its Drosophila melanogaster homolog as novel negative regulators of Notch signaling. USP12 silencing specifically interrupts Notch trafficking to the lysosomes and, as a consequence, leads to an increased amount of receptor at the cell surface and to a higher Notch activity. At the biochemical level, USP12 with its activator UAF1 deubiquitinate the nonactivated form of Notch in cell culture and in vitro. These results characterize a new level of conserved regulation of Notch signaling by the ubiquitin system.

The translation initiation factor 3f (eIF3f) exhibits a deubiquitinase activity regulating Notch activation.

Activation of the mammalian Notch receptor after ligand binding relies on a succession of events including metalloprotease-cleavage, endocytosis, monoubiquitination, and eventually processing by the gamma-secretase, giving rise to a soluble, transcriptionally active molecule. The Notch1 receptor was proposed to be monoubiquitinated before its gamma-secretase cleavage; the targeted lysine has been localized to its submembrane domain. Investigating how this step might be regulated by a deubiquitinase (DUB) activity will provide new insight for understanding Notch receptor activation and downstream signaling. An immunofluorescence-based screening of an shRNA library allowed us to identify eIF3f, previously known as one of the subunits of the translation initiation factor eIF3, as a DUB targeting the activated Notch receptor. We show that eIF3f has an intrinsic DUB activity. Knocking down eIF3f leads to an accumulation of monoubiquitinated forms of activated Notch, an effect counteracted by murine WT eIF3f but not by a catalytically inactive mutant. We also show that eIF3f is recruited to activated Notch on endocytic vesicles by the putative E3 ubiquitin ligase Deltex1, which serves as a bridging factor. Finally, catalytically inactive forms of eIF3f as well as shRNAs targeting eIF3f repress Notch activation in a coculture assay, showing that eIF3f is a new positive regulator of the Notch pathway. Our results support two new and provocative conclusions: (1) The activated form of Notch needs to be deubiquitinated before being processed by the gamma-secretase activity and entering the nucleus, where it fulfills its transcriptional function. (2) The enzyme accounting for this deubiquitinase activity is eIF3f, known so far as a translation initiation factor. These data improve our knowledge of Notch signaling but also open new avenues of research on the Zomes family and the translation initiation factors.

Involvement of the Notch pathway in the regulation of matrix metalloproteinase 13 and the dedifferentiation of articular chondrocytes in murine cartilage.

OBJECTIVE: To demonstrate the activation of the Notch signaling pathway during changes in the phenotype of chondrocytes in vitro, and to assess the influence of Notch on the production of chondrocyte markers. METHODS: Serial monolayer primary cultures of murine articular chondrocytes (MACs), as a model of chondrocyte dedifferentiation, were prepared. MACs were cultured with or without a Notch inhibitor and transfected with different Notch-expressing vectors. The Notch pathway and chondrocyte marker profiles were assessed by quantitative reverse transcription-polymerase chain reaction, immunoblotting, and immunocytochemistry. RESULTS: Successive passages of MACs resulted in a loss of type II collagen and aggrecan (chondrocyte differentiation markers), an increase in type I collagen (dedifferentiation marker), an increase in Notch ligands, and augmented target gene activity. The Notch inhibitor decreased the type II collagen protein content but had no effect on Col2a1 messenger RNA, while transfection with the constitutive active forms of the Notch1 receptor led to a decrease in type II collagen in transfected cells. In assays to investigate the mechanism of type II collagen breakdown, matrix metalloproteinase 13 (MMP-13) synthesis was regulated in a Notch-dependent manner, whereas MMP-2 synthesis was unchanged. CONCLUSION: The Notch signaling pathway is associated with decreased type II collagen production during the dedifferentiation of MACs in vitro. This may be correlated with the increase in MMP-13 production linked to activation of Notch.

Intracellular trafficking of Notch receptors and ligands.

The Notch pathway represents a highly conserved signaling network, which regulates the formation and maintenance of various organ systems along development and during adulthood. Direct cell-cell contacts between ligand- and receptor-expressing cells underlie activation of the Notch pathway. Notch signaling requires endocytosis in both signal emitting and receiving cells. Recent findings on the roles of a number of modulators show that they act either on the maintenance of an active receptor at the membrane, or on the production of active ligand, or on signal transduction after activation.

AIP4/Itch regulates Notch receptor degradation in the absence of ligand.

BACKGROUND: The regulation of Notch signaling heavily relies on ubiquitination events. Drosophila Su(dx), a member of the HECT family of ubiquitin-ligases, has been described as a negative regulator of Notch signaling, acting on the post-endocytic sorting of Notch. The mammalian ortholog of Su(dx), Itch/AIP4, has been shown to have multiple substrates, including Notch, but the precise events regulated by Itch/AIP4 in the Notch pathway have not been identified yet. METHODOLOGY/PRINCIPAL FINDINGS: Using Itch-/- fibroblasts expressing the Notch1 receptor, we show that Itch is not necessary for Notch activation, but rather for controlling the degradation of Notch in the absence of ligand. Itch is indeed required after the early steps of Notch endocytosis to target it to the lysosomes where it is degraded. Furthermore Itch/AIP4 catalyzes Notch polyubiquitination through unusual K29-linked chains. We also demonstrate that although Notch is associated with Itch/AIP4 in cells, their interaction is not detectable in vitro and thus requires either a post-translational modification, or a bridging factor that remains to be identified. CONCLUSIONS/SIGNIFICANCE: Taken together our results identify a specific step of Notch regulation in the absence of any activation and underline differences between mammalian and Drosophila Notch pathways.

Notch3 and IL-1beta exert opposing effects on a vascular smooth muscle cell inflammatory pathway in which NF-kappaB drives crosstalk.

Atherogenesis begins with the transfer of monocytes from the lumen to the intimal layer of arteries. The paracrine activity acquired by these monocytes shifts vascular smooth muscle cells from a contractile-quiescent to a secretory-proliferative phenotype, allowing them to survive and migrate in the intima. Transformed and relocated, they also start to produce and/or secrete inflammatory enzymes, converting them into inflammatory cells. Activation of the Notch pathway, a crucial determinant of cell fate, regulates some of the new features acquired by these cells as it triggers vascular smooth muscle cells to grow and inhibits their death and migration. Here, we evaluate whether and how the Notch pathway regulates the cell transition towards an inflammatory or de-differentiated state. Activation of the Notch pathway by the notch ligand Delta1, as well as overexpression of the active form of Notch3, prevents this phenomenon [initiated by interleukin 1beta (IL-1beta)], whereas inhibiting the Notch pathway enhances the transition. IL-1beta decreases the expression of Notch3 and Notch target genes. As shown by using an IkappaBalpha-mutated form, the decrease of Notch3 signaling elements occurs subsequent to dissociation of the NF-kappaB complex. These results demonstrate that the Notch3 pathway is attenuated through NF-kappaB activation, allowing vascular smooth muscle cells to switch into an inflammatory state.

Itch/AIP4 mediates Deltex degradation through the formation of K29-linked polyubiquitin chains.

Deltex (DTX) and AIP4 are the human orthologues of the Drosophila deltex and Suppressor of deltex, which have been genetically described as being antagonistically involved in the Notch signalling pathway. Both genes encode E3 ubiquitin ligases of the RING (Really interesting new gene)-H2 and HECT (Homologous to E6AP carboxyl terminus) families, respectively. In an attempt to understand the molecular basis of their genetic interactions, we studied the relationship between DTX and AIP4 in the absence of activation of the Notch pathway. We show here that both molecules interact and partially colocalize to endocytic vesicles, and that AIP4 targets DTX for lysosomal degradation. Furthermore, AIP4-generated polyubiquitin chains are mainly conjugated through lysine 29 of ubiquitin in vivo, indicating a link between this type of chain and lysosomal degradation.

Generation and characterization of mutant cell lines defective in gamma-secretase processing of Notch and amyloid precursor protein.

Several type I integral membrane proteins, such as the Notch receptor or the amyloid precursor protein, are cleaved in their intramembrane domain by a gamma-secretase enzyme, which is carried within a multiprotein complex. These cleavages generate molecules that are involved in intracellular or extracellular signaling. At least four transmembrane proteins belong to the gamma-secretase complex: presenilin, nicastrin, Aph-1, and Pen-2. It is still unclear whether these proteins are the only components of the complex and whether a unique complex is involved in the different gamma-secretase cleavage events. We have set up a genetic screen based on the permanent acquisition or loss of an antibiotic resistance depending on the presence of an active gamma-secretase able to cleave a Notch-derived substrate. We selected clones deficient in gamma-secretase activity using this screen on mammalian cells after random mutagenesis. We further analyzed two of these clones and identified previously undescribed mutations in the nicastrin gene. The first mutation abolishes nicastrin production, and the second mutation, a point mutation in the ectodomain, abolishes nicastrin maturation. In both cases, gamma-secretase activity on Notch and APP is impaired.