Eva Mortier

Syndecan-syntenin-ALIX regulates the biogenesis of exosomes

The biogenesis of exosomes, small secreted vesicles involved in signalling processes, remains incompletely understood. Here, we report evidence that the syndecan heparan sulphate proteoglycans and their cytoplasmic adaptor syntenin control the formation of exosomes. Syntenin interacts directly with ALIX through LYPX(n)L motifs, similarly to retroviral proteins, and supports the intraluminal budding of endosomal membranes. Syntenin exosomes depend on the availability of heparan sulphate, syndecans, ALIX and ESCRTs, and impact on the trafficking and confinement of FGF signals. This study identifies a key role for syndecan–syntenin–ALIX in membrane transport and signalling processes.

New PI(4,5)P2- and membrane proximal integrin–binding motifs in the talin head control β3-integrin clustering

A talin intermolecular interaction autoinhibits its own activation and regulates β3-integrin binding. When bound, β3-integrin undergoes structural alterations that prevent its β and α subunits from associating, maintaining β3-integrins clustering capability.

Nuclear speckles and nucleoli targeting by PIP2–PDZ domain interactions

PDZ (Postsynaptic density protein, Disc large, Zona occludens) domains are protein–protein interaction modules that predominate in submembranous scaffolding proteins. Recently, we showed that the PDZ domains of syntenin‐1 also interact with phosphatidylinositol 4,5‐bisphosphate (PIP2) and that this interaction controls the recruitment of the protein to the plasma membrane. Here we evaluate the general importance of PIP2–PDZ domain interactions. We report that most PDZ proteins bind weakly to PIP2, but that syntenin‐2, the closest homolog of syntenin‐1, binds with high affinity to PIP2 via its PDZ domains. Surprisingly, these domains target syntenin‐2 to nuclear PIP2 pools, in nuclear speckles and nucleoli. Targeting to these sites is abolished by treatments known to affect these PIP2 pools. Mutational and domain‐swapping experiments indicate that high‐affinity binding to PIP2 requires both PDZ domains of syntenin‐2, but that its first PDZ domain contains the nuclear PIP2 targeting determinants. Depletion of syntenin‐2 disrupts the nuclear speckles–PIP2 pattern and affects cell survival and cell division. These findings show that PIP2–PDZ domain interactions can directly contribute to subnuclear assembly processes.

Cutting edge: Dok-1 and Dok-2 adaptor molecules are regulated by phosphatidylinositol 5-phosphate production in T cells.

Downstream of tyrosine kinase (Dok) proteins Dok-1 and Dok-2 are involved in T cell homeostasis maintenance. Dok protein tyrosine phosphorylation plays a key role in establishing negative feedback loops of T cell signaling. These structurally related adapter molecules contain a pleckstrin homology (PH) domain generally acting as a lipid/protein-interacting module. We show that the presence of this PH domain is necessary for the tyrosine phosphorylation of Dok proteins and their negative functions in T cells. We find that Dok-1/Dok-2 PH domains bind in vitro to the rare phosphoinositide species, phosphatidylinositol 5-phosphate (PtdIns5P). Dok tyrosine phosphorylation correlates with PtdIns5P production in T cells upon TCR triggering. Furthermore, we demonstrate that PtdIns5P increase regulates Dok tyrosine phosphorylation in vivo. Together, our data identify a novel lipid mediator in T cell signaling and suggest that PH-PtdIns5P interactions regulate T cell responses.

The Pleckstrin Homology Domain of the Arf6-specific Exchange Factor EFA6 Localizes to the Plasma Membrane by Interacting with Phosphatidylinositol 4,5-Bisphosphate and F-actin

The Arf6-specific exchange factor EFA6 coordinates membrane trafficking with actin cytoskeleton remodeling. It localizes to the plasma membrane where it catalyzes Arf6 activation and induces the formation of actin-based membrane ruffles. We have shown previously that the pleckstrin homology (PH) domain of EFA6 was responsible for its membrane localization. In this study we looked for the partners of the PH domain at the plasma membrane. Mutations of the conserved basic residues suspected to be involved in the binding to phosphoinositides redistribute EFA6-PH to the cytosol. In addition, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) breakdown also leads to the solubilization of EFA6-PH. Direct binding measured by surface plasmon resonance gives an apparent affinity of ∼0.5 μm EFA6-PH for PI(4,5)P2. Moreover, we observed in vitro that the catalytic activity of EFA6 is strongly increased by PI(4,5)P2. These results indicate that the plasma membrane localization of EFA6-PH is based on its interaction with PI(4,5)P2, and this interaction is necessary for an optimal catalytic activity of EFA6. Furthermore, we demonstrated by fluorescence recovery after photobleaching and Triton X-100 detergent solubility experiments that in addition to the phophoinositides, EFA6-PH is linked to the actin cytoskeleton. We observed both in vivo and in vitro that EFA6-PH interacts directly with F-actin. Finally, we demonstrated that EFA6 could bind simultaneously filamentous actin and phospholipids vesicles. Our results explain how the exchange factor EFA6 via its PH domain could coordinate at the plasma membrane actin cytoskeleton organization with membrane trafficking.

The Postsynaptic Density 95/Disc-Large/Zona Occludens Protein Syntenin Directly Interacts with Frizzled 7 and Supports Noncanonical Wnt Signaling

Wnt signaling pathways are essential for embryonic patterning, and they are disturbed in a wide spectrum of diseases, including cancer. An unresolved question is how the different Wnt pathways are supported and regulated. We previously established that the postsynaptic density 95/disc-large/zona occludens (PDZ) protein syntenin binds to syndecans, Wnt coreceptors, and known stimulators of protein kinase C (PKC)alpha and CDC42 activity. Here, we show that syntenin also interacts with the C-terminal PDZ binding motif of several Frizzled Wnt receptors, without compromising the recruitment of Dishevelled, a key downstream Wnt-signaling component. Syntenin is coexpressed with cognate Frizzled during early development in Xenopus. Overexpression and down-regulation of syntenin disrupt convergent extension movements, supporting a role for syntenin in noncanonical Wnt signaling. Syntenin stimulates c-jun phosphorylation and modulates Frizzled 7 signaling, in particular the PKCalpha/CDC42 noncanonical Wnt signaling cascade. The syntenin-Frizzled 7 binding mode indicates syntenin can accommodate Frizzled 7-syndecan complexes. We propose that syntenin is a novel component of the Wnt signal transduction cascade and that it might function as a direct intracellular link between Frizzled and syndecans.

Syntenin, a syndecan adaptor and an Arf6 phosphatidylinositol 4,5-bisphosphate effector, is essential for epiboly and gastrulation cell movements in zebrafish

Epiboly, the spreading and the thinning of the blastoderm to cover the yolk cell and close the blastopore in fish embryos, is central to the process of gastrulation. Despite its fundamental importance, little is known about the molecular mechanisms that control this coordinated cell movement. By a combination of knockdown studies and rescue experiments in zebrafish (Danio rerio), we show that epiboly relies on the molecular networking of syntenin with syndecan heparan sulphate proteoglycans, which act as co-receptors for adhesion molecules and growth factors. Furthermore, we show that the interaction of syntenin with phosphatidylinositol 4,5-bisphosphate (PIP2) and with the small GTPase ADP-ribosylation factor 6 (Arf6), which regulate the endocytic recycling of syndecan, is necessary for epiboly progression. Analysis of the earliest cellular defects suggests a role for syntenin in the autonomous vegetal expansion of the yolk syncytial layer and the rearrangement of the actin cytoskeleton in extra-embryonic tissues, but not in embryonic cell fate determination. This study identifies the importance of the syntenin–syndecan–PIP2–Arf6 complex for the progression of fish epiboly and establishes its key role in directional cell movements during early development.

Evidence for a positive role of PtdIns5P in T-cell signal transduction pathways.

Phosphatidylinositol 5‐phosphate (PtdIns5P) is emerging as a potential lipid messenger involved in several cell types, from plants to mammals. Expression of IpgD, a PtdIns(4, 5)P2 4‐phosphatase induces Src kinase and Akt, but not ERK activation and enhances interleukin II promoter activity in T‐cells. Expression of a new PtdIns5P interacting domain blocks IpgD‐induced T‐cell activation and selective signaling molecules downstream of TCR triggering. Altogether, these data suggest that PtdIns5P may play a sensor function in setting the threshold of T‐cell activation and contributing to maintain T‐cell homeostasis.