Role of tetraspanin-syndecan-PDZ webs in the molecular and functional heterogeneity of extracellular vesicles – SynTEV

Our study was divided into 3 experimental parts. The first consisted of studying the impact of the deletion or mutation of 4 tetraspanins (CD9, CD63, CD81 and TSPAN6) on the intracellular trafficking of molecules (fluorescence microscopy) and on the composition of secreted EVs using a global approach (fractionation of EVs followed by proteomic studies). The second part aimed to establish a complete map of the direct interactions between the 4 tetraspanins, the 4 syndecans and the 150 human PDZ proteins by a genetic method (yeast two-hybrid) and to clarify the impact of ten PDZ proteins on the formation and composition of EVs (silencing method, imaging studies, fractionation and detection of specific proteins by Western blot). The third part aimed to clarify to what extent a disruption of the tetraspanins-syndecans-PDZ proteins networks could affect the capture of EVs by target cells (capture of fluorescent EVs followed by microscopy).

We have shown that (i) tetraspanins, major constituents of EVs, have little impact on their composition; (ii) syndecans would be reliable markers of small (nanometric) EVs; (iii) syndecans and tetraspanins cooperate for the release of EVs and (iv) PDZ proteins strongly control both the composition and capture of EVs, the latter by regulating the heparan sulfate chains which substitute the syndecans. This study allowed us to deepen our fundamental molecular knowledge on the biology of EVs and therefore to open the field to a more rational approach of their use.

Extracellular vesicles (EVs) are organelles controlling cell–cell communication in health and disease. Our understanding of the mechanisms regulating the heterogeneity of their composition and their selective uptake by recipient cells is limited. This impairs their rational use in diagnostics and therapeutics. This work unveiled paradigms governing EV exchange, as well as the biology of syndecans, tetraspanins, and PDZ proteins. These are fundamental findings that can now be exploited for the use of EVs as source of biomarkers or EV-based therapeutics.

Extracellular vesicles (EVs), including exosomes, transport complex information (lipids, proteins, nucleic acids). EVs participate in the regulation of multiple physio-pathological processes including immunity, angiogenesis, cancer, cardio-vascular diseases and neurodegeneration. The use of EVs for biomarker discovery and as vehicles or targets of therapies is currently under intense investigation. Yet poor knowledge of the molecular mechanisms controlling EV diversity precludes rational EV-based interventions.
Pioneer work and recent findings of our laboratories converge on the likeliness of an intimate interconnection of tetraspanins, syndecans and PDZ proteins that is crucial for EV biology. Our working hypothesis is that webs under the control of these scaffold molecules dictate the formation, the composition, the targeting and the function of EVs. Here we will identify direct molecular interactions governing the formation of these webs, and analyse by gain and loss-of-function studies how they impact on EV formation, heterogeneity, composition, targeting efficiency and functional effects on recipient cells. In the course of our work, we will also generate antibodies and validate flow cytometry based methods to quantify EV secretion.
This work will help clarifying presently poorly understood fundamental aspects of cell signalling and communication and potentially provide knowledge for rational design of innovative therapeutic intervention. Because of the potential of the project, we also included a formal task for the evaluation and protection of intellectual property.