Downregulation of stromal syntenin sustains AML development.

The crosstalk between cancer and stromal cells plays a critical role in tumor progression. Syntenin is a small scaffold protein involved in the regulation of intercellular communication that is emerging as a target for cancer therapy. Here, we show that certain aggressive forms of acute myeloid leukemia (AML) reduce the expression of syntenin in bone marrow stromal cells (BMSC). Stromal syntenin deficiency, in turn, generates a pro-tumoral microenvironment. From serial transplantations in mice and co-culture experiments, we conclude that syntenin-deficient BMSC stimulate AML aggressiveness by promoting AML cell survival and protein synthesis. This pro-tumoral activity is supported by increased expression of endoglin, a classical marker of BMSC, which in trans stimulates AML translational activity. In short, our study reveals a vicious signaling loop potentially at the heart of AML-stroma crosstalk and unsuspected tumor-suppressive effects of syntenin that need to be considered during systemic targeting of syntenin in cancer therapy.

Vitamin D Modulates Lipid Composition of Adipocyte-Derived Extracellular Vesicles Under Inflammatory Conditions.

SCOPE: Adipocyte-derived extracellular vesicles (AdEVs) convey lipids that can play a role in the energy homeostasis. Vitamin D (VD) has been shown to limit the metabolic inflammation as it decreases inflammatory markers expression in adipose tissue (AT). However, VD effect on adipocytes-derived EVs has never been investigated. METHODS AND RESULTS: Thus, the aim of this study is to evaluate the AdEVs lipid composition by LC-MS/MS approach in 3T3-L1 cells treated with VD or/and pro-inflammatory factor (tumor necrosis factor α [TNFα]). Among all lipid species, four are highlighted (glycerolipids, phospholipids, lysophospholipids, and sphingolipids) with a differential content between small (sEVs) and large EVs (lEVs). This study also observes that VD alone modulates EV lipid species involved in membrane fluidity and in the budding of membrane. EVs treated with VD under inflammatory conditions have different lipid profiles than the control group, which is more pronounced in lEVs. Indeed, 25 lipid species are significantly modulated in lEVs, compared with only seven lipid species in sEVs. CONCLUSIONS: This study concludes that VD, alone or under inflammatory conditions, is associated with specific lipidomic signature of sEVs and lEVs. These observations reinforce current knowledge on the anti-inflammatory effect of VD.

A Journey on Extracellular Vesicles for Matrix Metalloproteinases: A Mechanistic Perspective.

Matrix metalloproteinases (MMPs) are key players in matrix remodeling and their function has been particularly investigated in cancer biology. Indeed, through extracellular matrix (ECM) degradation and shedding of diverse cell surface macromolecules, they are implicated in different steps of tumor development, from local expansion by growth to tissue invasion and metastasis. Interestingly, MMPs are also components of extracellular vesicles (EVs). EVs are membrane-limited organelles that cells release in their extracellular environment. These "secreted" vesicles are now well accepted players in cell-to-cell communication. EVs have received a lot of interest in recent years as they are also envisioned as sources of biomarkers and as potentially outperforming vehicles for the delivery of therapeutics. Molecular machineries governing EV biogenesis, cargo loading and delivery to recipient cells are complex and still under intense investigation. In this review, we will summarize the state of the art of our knowledge about the molecular mechanisms implicated in MMP trafficking and secretion. We focus on MT1-MMP, a major effector of invasive cell behavior. We will also discuss how this knowledge is of interest for a better understanding of EV-loading of MMPs. Such knowledge might be of use to engineer novel strategies for cancer treatment. A better understanding of these mechanisms could also be used to design more efficient EV-based therapies.

[Tetraspanins and syndecans: Partners in crime for 'dealing' exosomes?] / Tétraspanines et syndécanes - Complices dans le « trafic ¼ des exosomes ?

Exosomes are small extracellular vesicles derived from endosomal compartments. The molecular mechanisms supporting the biology of exosomes, from their biogenesis to their internalization by target cells, rely on 'dedicated' membrane proteins. These mechanisms of action need to be further clarified. This will help to better understand how exosome composition and heterogeneity are established. This would also help to rationalize their use as source of biomarkers and therapeutic tools. Here we discuss how syndecans and tetraspanins, two families of membrane scaffold proteins, cooperate to regulate different steps of exosome biology.

TITLE: Tétraspanines et syndécanes - Complices dans le « trafic ¼ des exosomes ? ABSTRACT: Les exosomes sont de petites vésicules extracellulaires qui sont produites dans des compartiments endosomaux. Les mécanismes moléculaires sur lesquels reposent la biologie des exosomes, de leur biogenèse à leur internalisation par les cellules cibles, font notamment appel à des protéines membranaires particulières. Ces mécanismes méritent d'être clarifiés, afin de mieux comprendre la complexité de la composition des exosomes et de rationaliser leur utilisation comme biomarqueurs ou comme outils thérapeutiques. Nous discutons ici comment les syndécanes et les tétraspanines, deux familles de protéines d'échafaudage, coopèrent pour réguler les différentes étapes de la biologie des exosomes.

EFA6A, an exchange factor for Arf6, regulates early steps in ciliogenesis.

Ciliogenesis is a coordinated process initiated by the recruitment and fusion of pre-ciliary vesicles at the distal appendages of the mother centriole through mechanisms that remain unclear. Here, we report that EFA6A (also known as PSD), an exchange factor for the small G protein Arf6, is involved in early stage of ciliogenesis by promoting the fusion of distal appendage vesicles forming the ciliary vesicle. EFA6A is present in the vicinity of the mother centriole before primary cilium assembly and prior to the arrival of Arl13B-containing vesicles. During ciliogenesis, EFA6A initially accumulates at the mother centriole and later colocalizes with Arl13B along the ciliary membrane. EFA6A depletion leads to the inhibition of ciliogenesis, the absence of centrosomal Rab8-positive structures and the accumulation of Arl13B-positive vesicles around the distal appendages. Our results uncover a novel fusion machinery, comprising EFA6A, Arf6 and Arl13B, that controls the coordinated fusion of ciliary vesicles docked at the distal appendages of the mother centriole.

Tetraspanin-6 negatively regulates exosome production.

Exosomes, extracellular vesicles (EVs) of endosomal origin, emerge as master regulators of cell-to-cell signaling in physiology and disease. Exosomes are highly enriched in tetraspanins (TSPNs) and syndecans (SDCs), the latter occurring mainly in proteolytically cleaved form, as membrane-spanning C-terminal fragments of the proteins. While both protein families are membrane scaffolds appreciated for their role in exosome formation, composition, and activity, we currently ignore whether these work together to control exosome biology. Here we show that TSPN6, a poorly characterized tetraspanin, acts as a negative regulator of exosome release, supporting the lysosomal degradation of SDC4 and syntenin. We demonstrate that TSPN6 tightly associates with SDC4, the SDC4-TSPN6 association dictating the association of TSPN6 with syntenin and the TSPN6-dependent lysosomal degradation of SDC4-syntenin. TSPN6 also inhibits the shedding of the SDC4 ectodomain, mimicking the effects of matrix metalloproteinase inhibitors. Taken together, our data identify TSPN6 as a regulator of the trafficking and processing of SDC4 and highlight an important physical and functional interconnection between these membrane scaffolds for the production of exosomes. These findings clarify our understanding of the molecular determinants governing EV formation and have potentially broad impact for EV-related biomedicine.

Syntenin mediates SRC function in exosomal cell-to-cell communication.

The cytoplasmic tyrosine kinase SRC controls cell growth, proliferation, adhesion, and motility. The current view is that SRC acts primarily downstream of cell-surface receptors to control intracellular signaling cascades. Here we reveal that SRC functions in cell-to-cell communication by controlling the biogenesis and the activity of exosomes. Exosomes are viral-like particles from endosomal origin that can reprogram recipient cells. By gain- and loss-of-function studies, we establish that SRC stimulates the secretion of exosomes having promigratory activity on endothelial cells and that syntenin is mandatory for SRC exosomal function. Mechanistically, SRC impacts on syndecan endocytosis and on syntenin-syndecan endosomal budding, upstream of ARF6 small GTPase and its effector phospholipase D2, directly phosphorylating the conserved juxtamembrane DEGSY motif of the syndecan cytosolic domain and syntenin tyrosine 46. Our study uncovers a function of SRC in cell-cell communication, supported by syntenin exosomes, which is likely to contribute to tumor-host interactions.

Morphological and Functional Characterization of the Ciliary Pocket by Electron and Fluorescence Microscopy.

In many vertebrate cell types, the proximal part of the primary cilium is positioned within an invagination of the plasma membrane known as the ciliary pocket. Recent evidence points to the conclusion that the ciliary pocket comprises a unique site for exocytosis and endocytosis of ciliary proteins, which regulates the spatiotemporal trafficking of receptors into and out of the cilium to control its sensory function. In this chapter, we provide methods based on electron microscopy, 3D reconstruction of fluorescence images as well as live cell imaging suitable for investigating processes associated with endocytosis at the ciliary pocket.

Proteomic peptide phage display uncovers novel interactions of the PDZ1-2 supramodule of syntenin.

Syntenin has crucial roles in cell adhesion, cell migration and synaptic transmission. Its closely linked postsynaptic density-95, discs large 1, zonula occludens-1 (PDZ) domains typically interact with C-terminal ligands. We profile syntenin PDZ1-2 through proteomic peptide phage display (ProP-PD) using a library that displays C-terminal regions of the human proteome. The protein recognizes a broad range of peptides, with a preference for hydrophobic motifs and has a tendency to recognize cryptic internal ligands. We validate the interaction with nectin-1 through orthogonal assays. The study demonstrates the power of ProP-PD as a complementary approach to uncover interactions of potential biological relevance.

Arl13b and the exocyst interact synergistically in ciliogenesis.

Arl13b belongs to the ADP-ribosylation factor family within the Ras superfamily of regulatory GTPases. Mutations in Arl13b cause Joubert syndrome, which is characterized by congenital cerebellar ataxia, hypotonia, oculomotor apraxia, and mental retardation. Arl13b is highly enriched in cilia and is required for ciliogenesis in multiple organs. Nevertheless, the precise role of Arl13b remains elusive. Here we report that the exocyst subunits Sec8, Exo70, and Sec5 bind preferentially to the GTP-bound form of Arl13b, consistent with the exocyst being an effector of Arl13b. Moreover, we show that Arl13b binds directly to Sec8 and Sec5. In zebrafish, depletion of arl13b or the exocyst subunit sec10 causes phenotypes characteristic of defective cilia, such as curly tail up, edema, and abnormal pronephric kidney development. We explored this further and found a synergistic genetic interaction between arl13b and sec10 morphants in cilia-dependent phenotypes. Through conditional deletion of Arl13b or Sec10 in mice, we found kidney cysts and decreased ciliogenesis in cells surrounding the cysts. Moreover, we observed a decrease in Arl13b expression in the kidneys from Sec10 conditional knockout mice. Taken together, our results indicate that Arl13b and the exocyst function together in the same pathway leading to functional cilia.

Syntenin controls migration, growth, proliferation, and cell cycle progression in cancer cells.

The scaffold protein syntenin abounds during fetal life where it is important for developmental movements. In human adulthood, syntenin gain-of-function is increasingly associated with various cancers and poor prognosis. Depending on the cancer model analyzed, syntenin affects various signaling pathways. We previously have shown that syntenin allows syndecan heparan sulfate proteoglycans to escape degradation. This indicates that syntenin has the potential to support sustained signaling of a plethora of growth factors and adhesion molecules. Here, we aim to clarify the impact of syntenin loss-of-function on cancer cell migration, growth, and proliferation, using cells from various cancer types and syntenin shRNA and siRNA silencing approaches. We observed decreased migration, growth, and proliferation of the mouse melanoma cell line B16F10, the human colon cancer cell line HT29 and the human breast cancer cell line MCF7. We further documented that syntenin controls the presence of active ß1 integrin at the cell membrane and G1/S cell cycle transition as well as the expression levels of CDK4, Cyclin D2, and Retinoblastoma proteins. These data confirm that syntenin supports the migration and growth of tumor cells, independently of their origin, and further highlight the attractiveness of syntenin as potential therapeutic target.

Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2.

Exosomes are small vesicles that are secreted by cells and act as mediators of cell to cell communication. Because of their potential therapeutic significance, important efforts are being made towards characterizing exosomal contents. However, little is known about the mechanisms that govern exosome biogenesis. We have recently shown that the exosomal protein syntenin supports exosome production. Here we identify the small GTPase ADP ribosylation factor 6 (ARF6) and its effector phospholipase D2 (PLD2) as regulators of syntenin exosomes. ARF6 and PLD2 affect exosomes by controlling the budding of intraluminal vesicles (ILVs) into multivesicular bodies (MVBs). ARF6 also controls epidermal growth factor receptor degradation, suggesting a role in degradative MVBs. Yet ARF6 does not affect HIV-1 budding, excluding general effects on Endosomal Sorting Complexes Required for Transport. Our study highlights a novel pathway controlling ILV budding and exosome biogenesis and identifies an unexpected role for ARF6 in late endosomal trafficking.

Septins 2, 7 and 9 and MAP4 colocalize along the axoneme in the primary cilium and control ciliary length.

Septins are a large, evolutionarily conserved family of GTPases that form hetero-oligomers and interact with the actin-based cytoskeleton and microtubules. They are involved in scaffolding functions, and form diffusion barriers in budding yeast, the sperm flagellum and the base of primary cilia of kidney epithelial cells. We investigated the role of septins in the primary cilium of retinal pigmented epithelial (RPE) cells, and found that SEPT2 forms a 1:1:1 complex with SEPT7 and SEPT9 and that the three members of this complex colocalize along the length of the axoneme. Similar to observations in kidney epithelial cells, depletion of cilium-localized septins by siRNA-based approaches inhibited ciliogenesis. MAP4, which is a binding partner of SEPT2 and controls the accessibility of septins to microtubules, was also localized to the axoneme where it appeared to negatively regulate ciliary length. Taken together, our data provide new insights into the functions and regulation of septins and MAP4 in the organization of the primary cilium and microtubule-based activities in cells.

The ciliary pocket: a once-forgotten membrane domain at the base of cilia.

The PC (primary cilium) is present on most cell types in both developing and adult tissues in vertebrates. Despite multiple reports in the 1960s, the PC was almost forgotten for decades by most of the cell biology community, mainly because its function appeared enigmatic. This situation changed 10 years ago with the key discovery that this fascinating structure is the missing link between complex genetic diseases and key signalling pathways during development and tissue homoeostasis. A similar misfortune might have happened to an original membrane domain found at the base of PC in most cell types and recently termed the 'ciliary pocket'. A morphologically related structure has also been described at the connecting cilium of photoreceptors and at the flagellum in spermatids. Its organization is also reminiscent of the flagellar pocket, a plasma membrane invagination specialized in uptake and secretion encountered in kinetoplastid protozoa. The exact function of the ciliary pocket remains to be established, but the recent observation of endocytic activity coupled to the fact that vesicular trafficking plays important roles during ciliogenesis brought excitement in the ciliary community. Here, we have tried to decipher what this highly conserved membrane domain could tell us about the function and/or biogenesis of the associated cilium.

The AP-1 clathrin adaptor facilitates cilium formation and functions with RAB-8 in C. elegans ciliary membrane transport.

Clathrin adaptor (AP) complexes facilitate membrane trafficking between subcellular compartments. One such compartment is the cilium, whose dysfunction underlies disorders classified as ciliopathies. Although AP-1mu subunit (UNC-101) is linked to cilium formation and targeting of transmembrane proteins (ODR-10) to nematode sensory cilia at distal dendrite tips, these functions remain poorly understood. Here, using Caenorhabditis elegans sensory neurons and mammalian cell culture models, we find conservation of AP-1 function in facilitating cilium morphology, positioning and orientation, and microtubule stability and acetylation. These defects appear to be independent of IFT, because AP-1-depleted cells possess normal IFT protein localisation and motility. By contrast, disruption of chc-1 (clathrin) or rab-8 phenocopies unc-101 worms, preventing ODR-10 vesicle formation and causing misrouting of ODR-10 to all plasma membrane destinations. Finally, ODR-10 colocalises with RAB-8 in cell soma and they cotranslocate along dendrites, whereas ODR-10 and UNC-101 signals do not overlap. Together, these data implicate conserved roles for metazoan AP-1 in facilitating cilium structure and function, and suggest cooperation with RAB-8 to coordinate distinct early steps in neuronal ciliary membrane sorting and trafficking.

The ciliary pocket: an endocytic membrane domain at the base of primary and motile cilia.

Cilia and flagella are eukaryotic organelles involved in multiple cellular functions. The primary cilium is generally non motile and found in numerous vertebrate cell types where it controls key signalling pathways. Despite a common architecture, ultrastructural data suggest some differences in their organisation. Here, we report the first detailed characterisation of the ciliary pocket, a depression of the plasma membrane in which the primary cilium is rooted. This structure is found at low frequency in kidney epithelial cells (IMCD3) but is associated with virtually all primary cilia in retinal pigment epithelial cells (RPE1). Transmission and scanning electron microscopy, immunofluorescence analysis and videomicroscopy revealed that the ciliary pocket establishes closed links with the actin-based cytoskeleton and that it is enriched in active and dynamic clathrin-coated pits. The existence of the ciliary pocket was confirmed in mouse tissues bearing primary cilia (cumulus), as well as motile cilia and flagella (ependymal cells and spermatids). The ciliary pocket shares striking morphological and functional similarities with the flagellar pocket of Trypanosomatids, a trafficking-specialised membrane domain at the base of the flagellum. Our data therefore highlight the conserved role of membrane trafficking in the vicinity of cilia.

Targeting of beta-arrestin2 to the centrosome and primary cilium: role in cell proliferation control.

BACKGROUND: The primary cilium is a sensory organelle generated from the centrosome in quiescent cells and found at the surface of most cell types, from where it controls important physiological processes. Specific sets of membrane proteins involved in sensing the extracellular milieu are concentrated within cilia, including G protein coupled receptors (GPCRs). Most GPCRs are regulated by beta-arrestins, betaarr1 and betaarr2, which control both their signalling and endocytosis, suggesting that betaarrs may also function at primary cilium. METHODOLOGY/PRINCIPAL FINDINGS: In cycling cells, betaarr2 was observed at the centrosome, at the proximal region of the centrioles, in a microtubule independent manner. However, betaarr2 did not appear to be involved in classical centrosome-associated functions. In quiescent cells, both in vitro and in vivo, betaarr2 was found at the basal body and axoneme of primary cilia. Interestingly, betaarr2 was found to interact and colocalize with 14-3-3 proteins and Kif3A, two proteins known to be involved in ciliogenesis and intraciliary transport. In addition, as suggested for other centrosome or cilia-associated proteins, betaarrs appear to control cell cycle progression. Indeed, cells lacking betaarr2 were unable to properly respond to serum starvation and formed less primary cilia in these conditions. CONCLUSIONS/SIGNIFICANCE: Our results show that betaarr2 is localized to the centrosome in cycling cells and to the primary cilium in quiescent cells, a feature shared with other proteins known to be involved in ciliogenesis or primary cilium function. Within cilia, betaarr2 may participate in the signaling of cilia-associated GPCRs and, therefore, in the sensory functions of this cell "antenna".