Identification of a new protein signature of extracellular vesicles involved in embryonic development and pathology – ART-EVs

Differential mass spectrometry analysis of ART EVs

During our first round of mass spectrometry analysis of the content of Shh-containing vesicles (see preliminary results and Figure 2), we identified a total of 15 enriched proteins (Fold change > 2 and p value < 0.05) already identified in EV’s studies, and three more specific proteins that we defined as the ART proteins (Axl, Rab18 and TMED10). This preliminary experiment however was not sensitive enough to detect the whole content of the Shh-containing vesicles. Thus, a deep characterization of the protein content of ART EVs will be performed again using a latest generation, high sensitivity nanoLC-MS/MS coupling, including prior sample prefractionation if required to increase the proteome coverage towards its exhaustive characterisation. We initially compared Shh pull-down vesicles to irrelevant IgG pull-down. Here we propose to design a differential experiment to compare the protein content of ART EVs (Shh pull-down) to the content of conventional exosomes (CD63 pull-down) using a global quantitative label-free approach.

Origin and nature of the Shh-containing ART EVs

To determine whether Shh-containing ART EVs are budding into intracellular compartments or at the PM, we propose to perform ultrastructural studies using immuno-electron microscopy to detect the budding sites of Shh-positive vesicles. We have good anti-Shh antibodies that we successfully tested for EM applications. To confirm the identity of the Shh-loaded vesicles as ART EVs we will also perform double immuno-EM labeling. In the case where none of the 5 anti ART EVs antibodies available in Gaudin team are working by immuno-EM, we will transfect a Axl-EGFP contruct and use an anti-EGFP antibody in combination with the Shh antibody to acertain that Shh-containing ART EVs are budding into MVB and/or at the PM.

As a complementary approach to the first two tasks, functional studies will be carried out in Drosophila melanogaster. The conservation of Hh activity at both the molecular, cellular and tissue level make drosophila an attractive model compared to other systems less easily manipulable genetically. The role of the ART proteins will be tested in two genetically tractable tissues, the larval WIDs and the pupal histoblasts. Any defects in Hh secretion or signalling can be easily measured through the use of signalling sensors and the immunochemical tools available in the Thérond laboratory. We previously established that the ESCRT machinery controls the secretion of EV-bound Hh in the imaginal discs12.

Although expressed in Hh producing cells, not all drosophila orthologues of the ART proteins are functionally well defined. One single conserved orthologue for TMED10 (which belongs to the p24 protein family) has previously been shown to be involved in the secretion of the Wnt morphogen53. Orthologues for Axl show mild conservation (35%), while a single conserved Rab18 orthologue is present.

We employed the retention using selective hooks (RUSH) system in cultured human foetal astrocyte cells and showed that newly-synthesised SHH trafficks through the classical biosynthetic secretory pathway. We found that the SHH-RUSH construct is functional, retaining signalling activity and is transported from the ER to the Golgi up to the plasma membrane (PM) within 45 min. Silencing TMED10 significantly delays Hh loading onto ERES and subsequent exit leading to significant Hh release defects, strongly suggesting that TMED10 is necessary for ER-to-Golgi transport of Hh, while SHH uses Rab6 vesicles for Golgi-to-cell surface trafficking13. TMED10 is a member of the p24 complex transmembrane proteins that act as a cargo receptor in the ER lumen for incorporation of secretory cargo molecules into transport vesicles. In the WID model, we demonstrated that the homologue of TMED10, Baiser (Bai), participates in Hh secretion and signalling in vivo. This work highlights the role of TMED10 in cargo-specific egress from the ER13.

Interestingly, TMED10 has been found associated to SHH-containing ART-EVs, a subtype of EVs displaying a very particular signature with the presence of Axl, Rab18 and TMED10, and found in the cerebrospinal fluid of a glioblastoma patient 14. One of the most frequently used markers to label EVs originating from MVBs is CD6315, although it is not a universal marker for specific EVs. Interestingly, the SHH ART- EVs are devoided of CD6314.

Although we elucidated the role of TMED10 in the initial steps of the SHH neosynthetic secretory pathway, the presence of TMED10 on SHH-containing EVs and its potential extracellular role are very intriguing. Indeed, TMED10 is a ER-Golgi resident protein that is usually not found in EVs. Our preliminary data however clearly indicated that the expression of SHH induces TMED10 loading onto EVs. When, how, and why this happens remains to be resolved. The identification of this Hh-EV protein signature is therefore of general importance and strongly suggesting that specific markers for Hh-EVs in experimental model animals can be identified. Such knowledge will likely help to describe EVs not only based on composition, but also from a functional perspective.

Knowledge on the molecular mechanisms that regulate cellular homeostasis and organ function is lacking and this is critical to understand, and hopefully manipulate, the basic processes of adult tissue homeostasis and injury repair. More specifically, our knowledge about how cells exchange informations is still preliminary and models are highly debated. One important signal which is involved in cell homeostasis and tissue organization is the Hh secreted molecule. Understanding how this signal is exchanged between cells will likely help to understand mechanism of cell-cell interaction in general. Not only the Hh pathway is a major regulator of embryonic development (including nervous system development) but recent evidence indicates that the Hh pathway continues to function in adult tissue, normal as well as diseased, by regulating both cell proliferation and the production of growth and angiogenic factors. This dual ability has great potential for the regeneration of cells in adult life. The main objectives of this project are to elucidate the mechanisms regulating cell-cell interactions through the study of Hh secretion Hh and to explore how it is regulated in different pathologies.