Targeting of β-Arrestin2 to the Centrosome and Primary Cilium: Role in Cell Proliferation Control

International audienceBackground: 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 b-arrestins, barr1 and barr2, which control both their signalling and endocytosis, suggesting that barrs may also function at primary cilium.Methodology/Principal Findings: In cycling cells, βarr2 was observed at the centrosome, at the proximal region of the centrioles, in a microtubule independent manner. However, βarr2 did not appear to be involved in classical centrosome-associated functions. In quiescent cells, both in vitro and in vivo, βarr2 was found at the basal body and axoneme of primary cilia. Interestingly, βarr2 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, βarrs appear to control cell cycle progression. Indeed, cells lacking βarr2 were unable to properly respond to serum starvation and formed less primary cilia in these conditions.Conclusions/Significance: Our results show that βarr2 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, βarr2 may participate in the signaling of cilia-associated GPCRs and, therefore, in the sensory functions of this cell “antenna”

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

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.journal article2014 Mar 182014 03 18importe

Redécouverte et caractérisation de la poche ciliaire, un domaine membranaire à la base du cil primaire

PARIS5-BU Méd.Cochin (751142101) / SudocSudocFranceF

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

Tétraspanines et syndécanes: Complices dans le « trafic » des exosomes ?

International audienceLes 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

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

International audienceThe primary cilium (PC) is present on most cell types in both developing and adult tissues in vertebrates. Despite multiple reports in the sixties, the PC was almost forgotten for decades by most of the cell biology community, mainly because its function appeared enigmatic. This situation changed ten 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 homeostasis. 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

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

International audienceIn 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

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 β1 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.status: publishe

Association of BTN2A1+microvesicles with overall survival and the role of γδ T cells as a potential therapeutic target in patients with hormone-sensitive metastatic prostate cancer.

International audience194 Background: Despite the recent progress in cancer management, metastatic prostate cancer remains incurable. In the context of immunotherapy failure, we need to identify new immune biomarkers, to improve patient therapy, and explore their potential as therapeutic target. To this end, we screened the prognostic value of soluble immune checkpoints in the plasma of metastatic prostate cancer patients at diagnosis, and depicted their role in anti-tumor activities. Methods: Patients with hormone naive metastatic prostate cancer were prospectively included in the NK-Prostate study (NCT02963155). Blood collection was performed before treatment and patients were followed for 5 years. We determined by ELISA assay plasma levels the soluble form of PD-L1, Pan-BTN3A, BTN3A1, BTN2A1 and BTLA. Large extracellular vesicles (MVs) were pelleted at 10 000g by sequential ultracentrifugation. Results: 66 patients were included in the NK-Prostate with a median follow-up of 42.6 months. Only plasma levels of BTN2A1 were associated with overall survival > 50 months. Patients with enriched sBTN2A1 (soluble form of BTN2A1) presented poorer overall survival (HR for death 2.91 95%CI 1.33 – 6.33, p=0.0072) and progression-free survival than low-level patients (HR for progression 2.73 95%CI 1.48 – 5.05, p=0.0012). Prostate cancer cell line produced sBTN2A1, with 40% associated with extracellular vesicles. High level of BTN2A1+ microvesicles (MVs) were identified by spectral cytometry in patient’ plasma but not in healthy volunteer plasma, and deep phenotyping revealed that B cell-derived BTN2A1+ MVs were associated with shorter overall survival (HR 4.22 95%CI 1.68 – 10.57, p=0.0021). To evaluate the impact of BTN2A1+ MVs on γδ T cells, we generated BTN2A1 KO MVs, BTN2A1 overexpressed MVs, and BTN2A1 mutant overexpressed MVs (that do not interact with γδ TCR). We demonstrated that BTN2A1 overexpressed MVs could specifically interact with γδ T cell; and impaired their proliferation and cytotoxicity against prostate cancer cell lines (LNCaP, DU145 and PC3). Conclusions: Our study shows the negative impact of B cell derived BTN2A1+ MVs in hormone naïve metastatic prostate cancer. Plasma MVs phenotyping is an easy-to-perform test, accessible for all patients. Our results highlight the role of γδ T cell in the anti-tumor immunity in prostate cancer, making them an interesting target for immunotherapy in this pathology. Clinical trial information: NCT02963155 . http://www.clinicaltrials.gov/ct2/show/NCT0296315