José Ignacio Valenzuela
email: jose-ignacio.valenzuela@curie.fr

Franck Perez
email: franck.perez@curie.fr

Localized intercellular transfer of ephrin-As by trans-endocytosis enables long-term signaling

Ephrins can elicit either contact-mediated cell-cell adhesion or repulsion, depending on the efficiency of removal of their ligand/receptor complexes from the cell surface, thus controlling tissue morphogenesis and oncogenic development. However, the dynamic of turnover of the newly assembled ephrin/Eph complexes during cell-cell interactions remains mostly unexplored. Here we show that ephrin-A1/EphA2 complexes are locally formed at the tip of filopodia, at cell-to-cell contacts. Clusters of ephrin-A1 from donor cells surf on filopodia associated to EphA2-bearing sub-domains of acceptor cells. Fulllength ephrin-A1 is transferred to acceptor cells by trans-endocytosis through a proteolysisindependent mechanism. Trans-endocytosed ephrin-A1 bound to its receptor enables signaling to be emitted from endo-lysosomes of acceptor cells. Localized trans-endocytosis of ephrin-A1 sustains contact-mediated repulsion on cancer cells. Our results uncover the essential role played by local concentration at the tip of filopodia and the trans-endocytosis of full-length ephrin to maintain long-lasting ephrin signaling.

Introduction

Cell contact-mediated repulsion is a basic mechanism of cell and axon navigation that underlies tissue boundary formation and patterning [START_REF] Batlle | Molecular mechanisms of cell segregation and boundary formation in development and tumorigenesis[END_REF]. The fundamental principle relies on the high-affinity interaction between a surface-bound repellent and its receptor on opposing cell. The repellent-receptor complexes form a physical tether that must be removed after cell-cell contact to allow cell detachment and repulsion. This can be achieved by two conceptually different mechanisms: i) regulated cleavage of the ligand-receptor complex [START_REF] Hattori | Regulated cleavage of a contactmediated axon repellent[END_REF][START_REF] Janes | Adam meets Eph: An ADAM substrate recognition module acts as a molecular switch for ephrin cleavage in trans[END_REF], ii) transendocytosis of the complete ligand-receptor cluster [START_REF] Gaitanos | Tiam-Rac signaling mediates transendocytosis of ephrin receptor EphB2 and is important for cell repulsion[END_REF][START_REF] Marston | Rac-dependent trans-endocytosis of ephrinBs regulates Eph-ephrin contact repulsion[END_REF][START_REF] Zimmer | EphB-ephrinB bi-directional endocytosis terminates adhesion allowing contact mediated repulsion[END_REF]. Eph receptors (EphRs) are the largest subfamily of tyrosine kinases receptors and together with ephrins, their membrane-bound ligands, constitute important cell guiding cues during development and oncogenesis [START_REF] Batlle | Molecular mechanisms of cell segregation and boundary formation in development and tumorigenesis[END_REF][START_REF] Boyd | Therapeutic targeting of EPH receptors and their ligands[END_REF][START_REF] Kania | Mechanisms of ephrin-Eph signalling in development, physiology and disease[END_REF]. EphRs activate signaling pathways that directly impact cytoskeleton regulating cell-cell interactions, guiding cell migration, neuronal pathfinding and tissue patterning [START_REF] Kania | Mechanisms of ephrin-Eph signalling in development, physiology and disease[END_REF]. EphRs are classified into two subfamilies; EphA and EphB, depending on the membrane anchoring nature of the ligand they preferentially bind, either glycophosphatidylinositol (GPI)-anchored ephrins (ephrin-As type) or transmembrane ephrins (ephrin-Bs type) respectively (Eph Nomenclature Committee, 1997). Ephrin's linkage to membranes facilitates its clustering, which is essential for the efficient receptor activation [START_REF] Davis | Ligands for EPH-related receptor tyrosine kinases that require membrane attachment or clustering for activity[END_REF][START_REF] Stein | Eph receptors discriminate specific ligand oligomers to determine alternative signaling complexes, attachment, and assembly responses[END_REF].

The membrane localization of the ligands provides to the ephrin-Eph pathway the unique property of transducing reverse signals into the cells that express the ligand, in addition to the forward signals in the cells that contain the EphRs [START_REF] Klein | Bidirectional modulation of synaptic functions by Eph/ephrin signaling[END_REF][START_REF] Pasquale | Eph-Ephrin Bidirectional Signaling in Physiology and Disease[END_REF].

Under the current paradigm of ephrin-mediated repulsion, ephrin-B/EphB cell tethers are removed from the cell surface by trans-endocytosis of the whole complex including portions of the surrounding membranes [START_REF] Marston | Rac-dependent trans-endocytosis of ephrinBs regulates Eph-ephrin contact repulsion[END_REF], by a mechanism that is bidirectional [START_REF] Gaitanos | Tiam-Rac signaling mediates transendocytosis of ephrin receptor EphB2 and is important for cell repulsion[END_REF][START_REF] Zimmer | EphB-ephrinB bi-directional endocytosis terminates adhesion allowing contact mediated repulsion[END_REF] and depends on the intracellular domains of both proteins and the Rac pathway [START_REF] Gaitanos | Tiam-Rac signaling mediates transendocytosis of ephrin receptor EphB2 and is important for cell repulsion[END_REF][START_REF] Marston | Rac-dependent trans-endocytosis of ephrinBs regulates Eph-ephrin contact repulsion[END_REF] role of the trans-endocytosis of ephrin to sustain long-lasting signaling from endomembranes.

Results

Synchronization of the secretion of EphA2 and ephrin-A1

To finely tune the delivery of EphA and ephrin-As to the PM we used the RUSH (Retention Using Selective Hooks) system (Boncompain et al., 2012). RUSH allows the synchronization of the secretory trafficking of a chosen protein using biotin-sensitive cargo retention in the ER (Fig. 1A). We focused on the trafficking of ephrin-A1 and its receptor EphA2. We used HeLa cells because their morphology allows to easily identify various cellular compartment and they recapitulate in vitro the cell signaling through filopodia that have been observed in vivo [START_REF] Lidke | Reaching out for signals: filopodia sense EGF and respond by directed retrograde transport of activated receptors[END_REF][START_REF] Moti | Wnt traffic from endoplasmic reticulum to filopodia[END_REF]. We performed simultaneous live-cell imaging of SBP-Cherry-ephrin-A1 and EphA2-EGFP-SBP. Both cargoes were efficiently retained in the ER and rapidly exported from it after biotin addition, reaching the Golgi apparatus (GA) in ~5 min (Fig. 1 C,D). We measured the fluorescence intensity of the cargoes at the GA over the time as an indication of their trafficking kinetic. We observed that both cargoes peak at ~15 min (Fig. 1C,D) before experiencing an intra-GA segregation. A difference in GA exit kinetic is also observed, with ephrin-A1 residing longer than EphA2 in the GA (Fig. 1C to E and video S1). This coincides with the sorting of EphA2 and ephrin-A1, which are partially segregated in post-Golgi vesicles in route to the PM (Fig. 1C,E). As expected, and as a control, SBP-EGFPephrin-A1 and SBP-Cherry-ephrin-A1 fully co-localized during their secretory transport (Fig. 1E and video S1).

We observed that ephrin-A1, but not EphA2 was concentrated in filopodia-associated subdomains of the PM upon arriving at the cell surface (Fig. 1F). It then seemed to be exported into neighboring cells at later time points. We imaged the trafficking of SBP-EGFP-ephrin-A1 in live-cells co-expressing a mCherry-tagged PM marker (PM-Cherry).

We observed that ephrin-A1 clusters were formed at the base or all along filopodia ~20 min after biotin addition, where they often localize at the tip before being exported from ligand producing cells later on (Fig. 1G-I and video S2).

Eprhin-As are sorted into signaling filopodia

This phenomenon was also observed for another GPI-anchored ephrin-A. ephrin-A5 (SBP-EGFP-ephrin-A5) was also targeted to, and released from, the tip of filopodia upon arriving at the cell surface (Fig. 2A, and video S3). This suggests that the localized secretion on filopodia may be a general behavior for ephrin-As. We tested if this was a common behavior of GPI-anchored proteins (GPI-APs). We used the RUSH system to co-express ephrin-A1 together with different GPI-APs: a fully synthetic one (SBP-Cherry-GPI) or the naturally occurring GPI-APs (SBP-Cherry-CD59 and SBP-Cherry-ALPP). While all the GPI-APs we analyzed were co-transported with ephrin-A1 at early stages, they were excluded from the ephrin-A1-specific clusters at the PM and were not exported from transfected cells (Fig.

2B and video S4).

Because ephrin-A1 clusters were secreted from actin-rich filopodia, as visualized with the actin-binding peptide F-tractin-EGFP [START_REF] Belin | Comparative analysis of tools for live cell imaging of actin network architecture[END_REF] (Fig. 2C), we questioned the role of actin dynamics in this process. We used cytochalasin-D to depolymerize actin in cells co-transfected with SBP-EGFP-ephrin-A1 and PM-Cherry. Live-cell imaging in control cells confirmed that, as observed above for SBP-EGFPephrin-A5, SBP-EGFP-ephrin-A1 clusters were only formed at the cell-cell interfaces (Fig. 2A andD , video S3 and S5).

Interestingly, single filopodia can support many rounds of ephrin-A1 export, with the clusters being subsequently detected into neighboring cells. Surprisingly, despite the decrease in number and length of filopodia observed after actin disorganization (Fig. 2F-G), ephrin-A1 clusters were still produced with timing comparable to control cells. However they were rarely found at the tips of cellular extension and their transfer to neighboring cells was strongly impaired (Fig. 2D-E and H, video S5). End-point quantifications of fixed cells showed a significant decrease in the number of transferred ephrin-A1 clusters to the surrounding cells after cytochalasin-D or latrunculin-A treatments (Fig. 2H). These data suggest that newly synthetized ephrin-As might be targeted to specialized signaling filopodia and that transport to surrounding cells is actin-dependent.

Cleavage-independent trans-endocytosis of ephrin-A1

Our experiments suggested that ephrin-A1 was transferred from ligand-producing cells (donor) towards acceptor cells, but it was still possible that these externalized clusters existed outside from neighboring cells. We thus evaluated whether externalized ephrin-A1 clusters were localized inside acceptor cells. We induced the delivery of SBP-EGFPephrin-A1 to the PM using the RUSH system, stopped the transport on ice and immunolabeled extracellular SBP-EGFP-ephrin-A1 in non-permeabilizing conditions.

Then we permeabilized the cells and stained the total SBP-EGFP-ephrin-A1. Clusters immunostained only after permeabilization thus corresponded to an intracellular pool of ephrin-A1. Such clusters were clearly detected inside acceptor cells, implicating that ephrin-A1 expressed on a donor filopodia underwent endocytosis into acceptor cells (Fig. 3A).

To explain how a membrane-attached ephrin-A1 could be transferred to neighboring cells we examined whether it was cleaved at the tip of filopodia by ADAMs. We synchronized the trafficking of SBP-Cherry-ephrin-A1 after inhibition of ADAMs with the broad spectrum inhibitor TAPI-1 [START_REF] Janes | Adam meets Eph: An ADAM substrate recognition module acts as a molecular switch for ephrin cleavage in trans[END_REF]. Surprisingly, the intercellular transfer of ephrin-A1 from filopodia remained unaffected under ADAM inhibition (Fig. 3B,C), suggesting that it can proceed independently of proteolytic shedding, in contrast to proposed models [START_REF] Greene | Spatial organization of EphA2 at the cell-cell interface modulates trans-endocytosis of ephrinA1[END_REF][START_REF] Hattori | Regulated cleavage of a contactmediated axon repellent[END_REF][START_REF] Janes | Adam meets Eph: An ADAM substrate recognition module acts as a molecular switch for ephrin cleavage in trans[END_REF][START_REF] Janes | Cytoplasmic relaxation of active Eph controls ephrin shedding by ADAM10[END_REF]. It was however still possible that inhibition was incomplete or that an unrelated protease was responsible for the cleavage. Thus, we developed a fluorescent assay to unambiguously determine whether full-length ephrin-As can undergo trogocytosis as reported for ephrin-Bs [START_REF] Gaitanos | Tiam-Rac signaling mediates transendocytosis of ephrin receptor EphB2 and is important for cell repulsion[END_REF][START_REF] Gong | Gulp1 controls Eph/ephrin trogocytosis and is important for cell rearrangements during development[END_REF][START_REF] Marston | Rac-dependent trans-endocytosis of ephrinBs regulates Eph-ephrin contact repulsion[END_REF][START_REF] Zimmer | EphB-ephrinB bi-directional endocytosis terminates adhesion allowing contact mediated repulsion[END_REF]. We engineered a fusion protein, which consists of ephrin-A1 tagged at its N-terminal extremity with SBP-mCherry and at its C-terminal end, just before the GPI-anchor sequence, by EGFP (SBP-Cherry-ephrin-A1-EGFP-GPI) (Fig. 3D-F). Depending on the mode of transfer of ephrin-A1, two possible output were expected: i) if ephrin-A1 was transferred to the acceptor cells as a product of its proteolytic cleavage, the clusters in the acceptor cells should be mCherry-positive and EGFP-negative, conversely ii) if ephrin-As were transferred without being cleaved, the clusters in the acceptor cells should be both mCherrypositive and EGFP-positive. Imaging synchronized transport of SBP-Cherry-ephrin-A1-EGFP-GPI to the cell surface, we consistently observed that clusters of ephrin-A1 positive for both mCherry and EGFP were trans-endocytosed from donor filopodia by the acceptor cells (Fig. 3D-F and video S6A). As a control, we inserted two consensus ADAM cleavage sites (2xADAMtide) [START_REF] Caescu | Active-site determinants of substrate recognition by the metalloproteinases TACE and ADAM10[END_REF] between SBP-mCherry and the N-terminal of ephrin-A1-EGFP-GPI (SBP-Cherry-2xADAMtide-ephrin-A1-EGFP-GPI) (Fig. S1A).

When SBP-Cherry-2xADAMtide-ephrin-A1-EGFP-GPI arrived to the PM, ADAMs induced the shedding of the SBP-Cherry portion and green, but not red, clusters were transferred to the acceptor cells (Fig. S1B-D). Importantly, the cleavage of SBP-Cherry-2xADAMtide-ephrin-A1-EGFP-GPI was efficiently reverted after ADAM inhibition with TAPI-1 or GI254023X (Fig. S1E-J).

Our data indicated that ephrin-A1 is trans-endocytosed in cleavage-independent manner. Two possible mechanisms were considered: the trans-endocytosis of ephrin-A1 may depend on the removal of the GPI-anchors (i.e. by phospholipase C), or full-length ephrin-A1 may be trans-endocytosed as a GPI-AP. To distinguish between these possibilities we designed a chimera in which we replaced the GPI-anchor sequence of SBP-Cherry-ephrin-A1 by the transmembrane domain of ephrin-B1 (EB1) followed by an EGFP in the cytoplasmic domain (SBP-Cherry-ephrin-A1-TMD-EB1-EGFP). If trans-endocytosis of ephrin-A1 depends on GPI-anchor cleavage, the ephrin chimera should stay bound to the PM of the donor cell and should not be transferred to the acceptor cells. On the contrary, if the whole ephrin-A undergoes trans-endocytosis, transfer may occur and the transendocytosed clusters should be mCherry-and EGFP-positive. Live-cell imaging revealed that clusters of SBP-Cherry-ephrin-A1-TMD-EB1-EGFP were pinched-off from the donor filopodia and trans-endocytosed as mCherry-and EGFP-positive clusters (Fig. 2G-I and video S6B). Our results thus demonstrate that ephrin-As can be transferred to neighboring cells as full-length proteins probably by trogocytosis.

Dynamin-dependent trans-endocytosis of ephrin-A1 from cytonemes

There are two main structures reported to connect donor and target cells through filopodiabased membrane bridges, namely cytonemes and tunneling nanotubes (TNTs) [START_REF] Sherer | Cytonemes and tunneling nanotubules in cellcell communication and viral pathogenesis[END_REF]. Cytonemes are specialized filopodia that connect distant cells through synaptic-like contacts, transporting signaling molecules, participating, for example, to establish a gradient of membrane bound morphogens in vivo [START_REF] Kornberg | Communicating by touch -neurons are not alone[END_REF].

Unlike cytonemes, TNTs are cell extensions that directly connect the cytoplasm of donor and acceptor cells, enabling the intercellular transfer of cytosolic content such as protein aggregates, endosomes and lysosomes [START_REF] Abounit | Tunneling nanotubes spread fibrillar α-synuclein by intercellular trafficking of lysosomes[END_REF][START_REF] Burtey | Intercellular transfer of transferrin receptor by a contact-, Rab8-dependent mechanism involving tunneling nanotubes[END_REF][START_REF] Gousset | Prions hijack tunnelling nanotubes for intercellular spread[END_REF][START_REF] Sherer | Cytonemes and tunneling nanotubules in cellcell communication and viral pathogenesis[END_REF]. In contrast to cytonemes, intercellular transfer of cargoes through TNTs is insensitive to endocytosis block (e.g. dynamin inhibition) [START_REF] Abounit | Tunneling nanotubes spread fibrillar α-synuclein by intercellular trafficking of lysosomes[END_REF][START_REF] Burtey | Intercellular transfer of transferrin receptor by a contact-, Rab8-dependent mechanism involving tunneling nanotubes[END_REF]. Thus, to differentiate between both mechanisms, we first inhibited dynamin-dependent endocytosis by pre-incubating SBP-EGFP-ephrin-A1 transfected cells and acceptor cells with the dynamin inhibitor Dyngo4A. Dyngo4A treatment efficiently inhibited endocytosis (as seen by transferrin uptake) as well as the intercellular transfer of SBP-EGFP-ephrin-A1 (Fig. 4A-C). We further blocked dynamindependent endocytosis specifically in acceptor cells by co-culturing SBP-EGFP-ephrin-A1 expressing cells with cells overexpressing either wild-type dynamin-1-CFP (Dynamin-1-WT) or the GTPase-defective/dominant-negative dynamin-1 (Dynamin-1-K44A). As expected, ephrin-A1 clusters were efficiently transferred through SBP-EGFP-ephrin-A1/Dynamin-1-WT interfaces (Fig. 4D and video S7). However, the internalization of ephrin-A1 clusters was strongly inhibited in Dynamin-1-K44A expressing cells, tending to accumulate at the donor/acceptor interface (Fig. 4D and video S7). These data indicate that ephrin-A1 is trans-endocytosed from cytoneme-like filopodia rather than transferred through TNTs.

We then analyzed the endocytic pathway followed by ephrin-A1 upon transfer into receiving cells. SBP-EGFP-ephrin-A1 expressing cells were mixed with cells expressing either wild-type or GTP-locked Rab5 mutant (mRFP-Rab5-WT or mCherry-Rab5-Q79L, respectively). Rab5 localizes at early endosomes and its GTP-bound form causes early endosome fusion. Importantly, mCherry-Rab5-Q79L expression in acceptor cells blocked ephrin-A1 inside Rab5-enlarged endosomes (Fig. 4E). Early endosomes-bearing transendocytosed ephrin-A1 mature to late endosomes, as visualized by the co-culture of SBP-Cherry-ephrin-A1 expressing cells with cells expressing Rab7 (EGFP-Rab7-WT) or the active GTPase-deficient mutant of Rab7 (EGFP-Rab7-Q76L) (Fig 4F). In contrast, transendocytosed ephrin-A1 is sorted apart from recycling endosomes, as indicated by the same type of experiments done using cells overexpressing Rab11 (EGFP-Rab11-WT) or the dominant negative GDP-bound form of Rab11 (DsRed-Rab11-S25N) (Fig. S2).

Localized trans-endocytosis of ephrin-A1 in complex with EphA2

We wondered whether the high local concentration of ephrin-A1 at filopodia tip might confine the ephrin signaling. We performed immunostaining after SBP-EGFP-ephrin-A1 secretion using an anti-phosphotyrosine antibody or an antibody that recognizes the active form of the EphARs (pEphA). We observed a strong co-localization between ephrin-A1 clusters and either phosphotyrosine signal or pEphA at the PM and inside the acceptor cells (Fig. 5A). Signaling started at the PM because when trans-endocytosis was blocked, activation of EphARs was still observed (Fig S3A). Signaling at the cell surface was actually linked to trans-endocytosis. Pharmacological inhibition of EphA2 signaling with Dasatinib [START_REF] Chang | Effects of dasatinib on EphA2 receptor tyrosine kinase activity and downstream signalling in pancreatic cancer[END_REF] efficiently impaired trans-endocytosis of ephrin-A1 (Fig. 5B and Fig. S3B). In these conditions, steady clusters of ephrin-A1 were observed at the interface between donor filopodia and acceptor cells (Fig. 5B-C).

To evaluate the dynamics of ephrin-A1/EphA2 signaling complexes, we co-cultured SBP-EGFP-ephrin-A1 expressing cells with EphA2-Cherry-SBP expressing cells and performed live-cell imaging upon trafficking synchronization. Under these conditions, EphA2-bearing cells extend filopodia towards the ephrin-A1-filopodia. We detected the local formation of high-order ligand/receptor clusters in subdomains of trans-interaction between donor and acceptor filopodia (Fig. 5D). Ephrin-A1/EphA2 complexes were preferentially transported through EphA2-containing filopodia towards the cell body of the acceptor cells where they underwent trans-endocytosis (Fig. 5D and video S17) although we sometimes observed clusters of EphA2Rs being pulled towards the cell body of ephrin-A1-producing cells (Fig. 5D and video S8). We questioned the trans-endocytosis of EphA2 by synchronizing the trafficking of extracellular-tagged SBP-EGFP-EphA2 co-cultured with cells transfected with SBP-Cherry-ephrin-A1. (Fig. 5E-F). We observed that a minor population of EphA2Rs might also undergo intercellular transfer.

Long-lasting signaling of trans-endocytosed ephrin-A1/EphA2 complexes in cancer cells

RUSH-based synchronization of transport was essential to reveal filopodia-dependent trans-endocytosis, however the intercellular transfer of ephrin-A1 to EphA2-expressing cells was massive at steady state (no hook expression) (video S9). This ruled-out possible artifacts due to trafficking synchronization. We then monitored transfer in cells known to use the ephrin-A1/EphA2 feedback loop. We used the highly aggressive breast cancer cell MDA-MB231, because they connect to the endothelia through filopodia promoting the formation of a metastatic niche in mice [START_REF] Connor | Physical nanoscale conduitmediated communication between tumour cells and the endothelium modulates endothelial phenotype[END_REF]. We co-cultured MDA-MB231 cells stably expressing SBP-EGFP-EphA2 (MDA-EphA2) with MDA-MB231 cells stably expressing SBP-Cherry-ephrin-A1 (MDA-ephrin-A1). Live imaging revealed that ephrin-A1 undergoes massive trans-endocytosis in a complex with the EphA2R. Intracellularly; ephrin-A1/EphA2 clusters accumulate into large and round endomembrane compartments of MDA-EphA2 cells (Fig. 6A). Ephrin-A1 was still detected inside those compartments more than 7 h after release from the ER. We analyzed whether MDA-MB231 cells use lysosomes for the long-term storage of ephrin-A1. We co-cultured donor SBP-Cherryephrin-A1 expressing cells with acceptor MDA-EphA2 cells and we labeled the lysosomes using the SiR-lysosome dye, which binds to the lysosomal proteinase cathepsin-D. We observed that ephrin-A1/EphA2 complexes are co-internalized and transported to large endo-lysosomal compartments (Fig. S4). To analyze whether sustained signaling accompanied this internalization, we co-cultured donor MDA-ephrin-A1 cells with wildtype MDA-MB231 cells used as acceptors. We labeled the lysosomal-associated membrane protein 1 (LAMP1) and active pEphARs. This experiment showed that trans-endocytosed ephrin-A1 was stored in lysosomes as signaling complexes. To better characterize the signaling compartment, we assessed for the acidity and degradative properties of the ephrin-A1-lysosomes by live-cell imaging using the pH-sensitive dye lysotracker and the fluorogenic protease substrate DQ-BSA respectively. We observed that trans-endocytosed ephrin-A1 is trafficked to acidic lysosomes, but just a minor population was found in degradative lysosome (Fig. S4). Interestingly, ephrin-A1-positive endo-lysosomes were slightly larger and concentrated a significantly higher amount of active EphARs than their ephrin-A1-negative counterpart (Fig. 6B-D).

In addition to protease-dependent cleavage, trans-endocytosis may thus be an alternative mechanism to allow cell separation and repulsion upon ephrin signaling. To measure contact-mediated cell repulsion we used a cell-confrontation assay [START_REF] Porazinski | EphA2 Drives the Segregation of Ras-Transformed Epithelial Cells from Normal Neighbors[END_REF].

It consists in a two-compartment cell culture insert separated by a fixed gap. After removal of the insert, cells migrate to fill the gap and opposing cells meet and establish cell-to-cell contacts. As a control, when two monolayers of MDA-ephrin-A1 cells were confronted, both populations stopped migrating upon contact (Fig. 7A,B), suggesting that no repulsion signal was emitted. In contrast, when acceptor MDA-EphA2 cells were confronted to donor MDA-ephrin-A1 cells, the acceptor MDA-EphA2 cells were repelled backward; meanwhile the MDA-ephrin-A1 cells persisted migrating forward (Fig. 7A,B). MDA-EphA2 repulsion correlated with the transfer of ephrin-A1 to acceptor cells, suggesting that trans-endocytosis of ephrin-A1 was used to remove the cell-cell tether formed by ephrin-A1 and EphA2.

Remarkably, when a MDA-EphA2 acceptor cell undergoes cell division short after ephrin-A1 trans-endocytosis, ephrin-A1 was inherited to the daughter cells inside an endomembrane compartment (Fig. 7C and video S10). To determine if activated EphARs can be inherited during cell division, MDA-MB231 cells labeled with a cell dye were seeded on gridded cell dishes and synchronized at the G2 phase of the cell cycle with the CDK1 inhibitor RO3306. Then MDA-ephrin-A1 cells were seeded on top of the acceptor cells in presence of biotin. Once the acceptor endosomes were filled with ephrin-A1, biotin and RO3306 were washed out and we imaged live the acceptor cells mitosis (Fig. 7D). We corroborated that trans-endocytosed ephrin-A1 is transmitted to daughter cells. When acceptor cells completed mitosis, we fixed them and co-stained with antibodies against LAMP-1 and pEphA. Importantly, we observed that around 60% of the inherited ephrin-A1 was localized into lysosomes and about 40% of inherited ephrin-A1-clusters contained pEphA (Fig. 7D-E), suggesting that a memory of ephrin signaling might be transmitted over generations.

Discussion

Membrane-bound ephrins and EphRs are ubiquitous signaling molecules present in most of cell types and regulating a myriad of biological processes. The localization and clustering of ephrins at the PM is required for the efficient receptor activation [START_REF] Davis | Ligands for EPH-related receptor tyrosine kinases that require membrane attachment or clustering for activity[END_REF][START_REF] Stein | Eph receptors discriminate specific ligand oligomers to determine alternative signaling complexes, attachment, and assembly responses[END_REF], restricting the ephrin/Eph communication to the cell vicinity at sites of cell-to-cell contact. However, there are some exceptions to this juxtacrine ephrin signaling.

Ephrin-A1 can be released in soluble forms by matrix metalloproteases activating an EphA2-dependent signaling at paracrine level [START_REF] Beauchamp | EphrinA1 Is Released in Three Forms from Cancer Cells by Matrix Metalloproteases[END_REF]Wykosky et al., 2008). Ectodomains of EphAs, EphBs and ephrin-Bs can also be released by proteolytic shedding, leading to signaling inhibition [START_REF] Barquilla | Eph receptors and ephrins: therapeutic opportunities[END_REF]. Additionally, cells can display EphAs, EphBs and ephrin-Bs on extracellular vesicles (EVs); which would expand the signaling to the endocrine level [START_REF] Gong | Exosomes mediate cell contactindependent ephrin-Eph signaling during axon guidance[END_REF]. Ephrin-As and -Bs may thus differ on their mechanisms of long-range communication.

Here we have uncovered a mechanism supporting long-range action for ephrin-As, namely cytonemes. Contacts produced by cytonemes are considered as a subtype of synaptic signaling [START_REF] Kornberg | Communicating by touch -neurons are not alone[END_REF]. Transport of ephrin-A on cytonemes brings several advantages: i) it allows ephrin-A to be concentrated at the tip of filopodia for efficient signaling; ii) the length of these filopodia enables the cells to sustain long-distance communication with EphARs displayed on acceptor PM; iii) concentration of the ligand at the tip of a very fine structure allows its efficient trans-endocytosis. This phenomenon, while massive at steady state, may have been overlooked so far. Synchronization of transport allowed generating a pulse that revealed this phenomenon.

The RUSH system enables the coordinated delivery of ephrins and EphRs to the PM, which is a powerful approach to the study of ephrin signaling and trans-endocytosis without the need for artificial clustering of soluble and non-physiological ligands. PM association endows ephrins with the particularity of transduce bidirectional signaling [START_REF] Pasquale | Eph-Ephrin Bidirectional Signaling in Physiology and Disease[END_REF].

The fact that a small proportion of EphA2Rs are trans-endocytosed in ephrin-A1 producing cells suggests that bidirectional signaling may take place at cytonemes. Interestingly, in sea urchin embryos a transmembrane ephrin has been observed to localize to cytonemes at the basal surface of ectodermal cells [START_REF] Krupke | Eph and Ephrin function in dispersal and epithelial insertion of pigmented immunocytes in sea urchin embryos[END_REF]. In zebrafish, the ephrin-B1/EphB3b signaling regulates cytonemal contacts between hepatoblast and the adjacent lateral plate mesoderm, coordinating liver morphogenesis and laterality [START_REF] Cayuso | EphrinB1/EphB3b Coordinate Bidirectional Epithelial-Mesenchymal Interactions Controlling Liver Morphogenesis and Laterality[END_REF]. In mammalian cells, myosin1b regulates the formation of filopodia at the ephrin-B1/EphB2 cellular interfaces, contributing to cell repulsion [START_REF] Prospéri | Myosin 1b functions as an effector of EphB signaling to control cell repulsion[END_REF]. Considering that ephrin-B/EphBR clusters are removed from the cell surface by trans-endocytosis [START_REF] Gaitanos | Tiam-Rac signaling mediates transendocytosis of ephrin receptor EphB2 and is important for cell repulsion[END_REF][START_REF] Marston | Rac-dependent trans-endocytosis of ephrinBs regulates Eph-ephrin contact repulsion[END_REF][START_REF] Zimmer | EphB-ephrinB bi-directional endocytosis terminates adhesion allowing contact mediated repulsion[END_REF], it is reasonable to propose that filopodia localization and trans-endocytosis are closely linked signal transduction processes.

Importantly, we show that removal of ephrin-A/EphA complexes is cleavage-independent.

This finding suggests an alternative to the accepted model of cell-tether removal and EphAR downregulation upon ephrin-A stimulation, which relies on the ADAM-dependent cleavage [START_REF] Hattori | Regulated cleavage of a contactmediated axon repellent[END_REF][START_REF] Janes | Adam meets Eph: An ADAM substrate recognition module acts as a molecular switch for ephrin cleavage in trans[END_REF][START_REF] Janes | Cytoplasmic relaxation of active Eph controls ephrin shedding by ADAM10[END_REF]. While it is still unclear whether trans-endocytosis has a distinct role from simple ephrin-A shedding, our results suggest that ADAMs might play an indirect role on cell-cell repulsion. For example, they may be used to regulate the shedding of E-cadherin, which have been observed on ephrin-B1/EphB boundaries [START_REF] Solanas | Cleavage of E-cadherin by ADAM10 mediates epithelial cell sorting downstream of EphB signalling[END_REF] or the shedding of EGF and further down-regulation of Ecadherin, as shown for ephrin-A1/EphA2 cell interfaces [START_REF] Kaplan | EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling[END_REF]. This would explain how the recruitment of ADAM10 to the ephrin-A/EphA interfaces can regulate repulsion independently of ephrin-A cleavage.

Ephrin-Bs also undergo bidirectional trogocytosis [START_REF] Gaitanos | Tiam-Rac signaling mediates transendocytosis of ephrin receptor EphB2 and is important for cell repulsion[END_REF][START_REF] Gong | Gulp1 controls Eph/ephrin trogocytosis and is important for cell rearrangements during development[END_REF][START_REF] Marston | Rac-dependent trans-endocytosis of ephrinBs regulates Eph-ephrin contact repulsion[END_REF][START_REF] Zimmer | EphB-ephrinB bi-directional endocytosis terminates adhesion allowing contact mediated repulsion[END_REF], suggesting that all sorts of ephrins may be subjected to it. Trans-endocytosis of ephrin-As depends on actin. Similarly, actin and Rac1 have been reported to be involved in trans-endocytosis of ephrin-Bs [START_REF] Gaitanos | Tiam-Rac signaling mediates transendocytosis of ephrin receptor EphB2 and is important for cell repulsion[END_REF][START_REF] Marston | Rac-dependent trans-endocytosis of ephrinBs regulates Eph-ephrin contact repulsion[END_REF]. Rac-1 has also been implicated in ephrin-A1-induced EphA2 endocytosis [START_REF] Zhuang | Regulation of EphA2 receptor endocytosis by SHIP2 lipid phosphatase via phosphatidylinositol 3-kinasedependent Rac1 activation[END_REF] and in ephrin-A5-induced EphA8 endocytosis during the development of the retinocollicuar topographic map in mice [START_REF] Yoo | EphA8-ephrinA5 signaling and clathrin-mediated endocytosis is regulated by Tiam-1, a Rac-specific guanine nucleotide exchange factor[END_REF][START_REF] Yoo | Endocytosis of EphA receptors is essential for the proper development of the retinocollicular topographic map[END_REF]. In neurons, ephrin-A2 induces Rac-1 activation, stimulating endocytosis in growth cones leading to their collapse [START_REF] Jurney | Rac1-mediated endocytosis during ephrin-A2-and semaphorin 3A-induced growth cone collapse[END_REF]. We have shown that ephrin-As interact with EphARs at filopodia. In embryonic retinal explants, ephrin-A2 and EphA4 colocalize in axons regulating the density of axonal interstitial filopodia [START_REF] Fiore | Regulation of axonal EphA4 forward signaling is involved in the effect of EphA3 on chicken retinal ganglion cell axon growth during retinotectal mapping[END_REF]. Early after birth, EphA4 receptors are located at the tip of axonal and dendritic filopodia in rat hippocampus [START_REF] Tremblay | Developmental course of EphA4 cellular and subcellular localization in the postnatal rat hippocampus[END_REF]. These data support the role of filopodia on ephrin-A-signaling in vivo.

Trogocytosed clusters of ephrin-A1 often contain small amounts of donor PM. In contrast to the shedding mechanism, trans-endocytosis allows the transfer of a membrane-attached ligand, hence keeping the ephrin in an oligomeric state and enabling efficient EphR activation. This would explain how trans-endocytosed ephrins are able to emit long-lasting signaling from lysosomes. Trans-endocytosed ephrin-A1/EphA2 complexes are transported using Rab5-and Rab7-endosomes (Fig. 7F). Similarly, fibroblast growth factor (FGF) forms a complex with cytoneme-localized FGF receptors and Rab5, which is retrogradely transported to the cell body [START_REF] Koizumi | RhoD activated by fibroblast growth factor induces cytoneme-like cellular protrusions through mDia3C[END_REF]. EGF (epidermal growth factor)/ErbB1 complexes are also endocytosed at the base of filopodia [START_REF] Lidke | Reaching out for signals: filopodia sense EGF and respond by directed retrograde transport of activated receptors[END_REF], suggesting that different cytoneme-related signaling molecules may follow similar endocytic pathways.

Ephrin-A1-dependent EphA2 activation and internalization leads to a transient inactivation of ERK and Akt pathways [START_REF] Sabet | Ubiquitination switches EphA2 vesicular traffic from a continuous safeguard to a finite signalling mode[END_REF]. Ephrin-A1-dependent Akt inhibition captures EGFR in Rab5 endosomes, therefore suppressing the pro-migratory Akt signaling from the PM while preserving proliferative endosomal ERK activity [START_REF] Stallaert | Contact inhibitory Eph signaling suppresses EGF-promoted cell migration by decoupling EGFR activity from vesicular recycling[END_REF].

In this context, cells with reduced cell-cell contact and therefore reduced ephrin signaling (e.g. at tissue boundaries) would increase their exploratory response to EGF, while cells located deeper into the tissue, would retain their proliferative response and fill the empty space despite their massive cell-cell contacts [START_REF] Stallaert | Contact inhibitory Eph signaling suppresses EGF-promoted cell migration by decoupling EGFR activity from vesicular recycling[END_REF]. A consequence of the endosomal sorting process is that it allows the transmission of ephrin-A/EphA complexes to the cellular progeny. This is possible because lysosomes are partitioned during cell division [START_REF] Bergeland | Mitotic partitioning of endosomes and lysosomes[END_REF]. Ephrin-loaded lysosomes may act then as long-term signaling unit, endowing the cell with a kind of memory. However, it will be essential to demonstrate that, in contrast to cleavage-dependent ephrin-A regulation, trans-endocytosis and complex storage in lysosomes provide a memory of signaling in vivo.

Long-distance communication through cytonemes, followed by storage in endomembranes may also promote the release of EphARs on EVs, switching to a cell contact-independent signaling modality (Fig. 7G). EphA2 on EVs may then both, activate reverse ephrinsignaling or titer down free ephrin-As on remote tissues. Because TNTs also participate in the intercellular transport of lysosomes [START_REF] Abounit | Tunneling nanotubes spread fibrillar α-synuclein by intercellular trafficking of lysosomes[END_REF] they may help to transfer signaling lysosomes between cells. This might be especially important in tumor cells, in which specialized filopodia, were proposed to configure a network that provides resistance to therapies [START_REF] Osswald | Brain tumour cells interconnect to a functional and resistant network[END_REF]. Notably, EphA2 overexpression decreases the sensitivity of breast cancer to tamoxifen and trastuzumab [START_REF] Lu | EphA2 overexpression decreases estrogen dependence and tamoxifen sensitivity[END_REF][START_REF] Zhuang | Elevation of receptor tyrosine kinase EphA2 mediates resistance to trastuzumab therapy[END_REF]. It would be important to know if the potential transfer of lysosomes containing active EphA2Rs might spread a resistance to otherwise sensitive cells. A pending challenge is to decipher the role in both normal and pathological development of the pathway described here for ephrins, from transport and concentration on cytoneme to trans-endocytosis and long-term signaling. Such a pathway may also be at work for other signaling routes linking cell-cell adhesion to signaling and may represent a signature of cytoneme-based signaling. Trans-illumination images (gray). Right panels show a similar experiment done using MDA-EGFP-EphA2 (green) confronted to MDA-Cherry-ephrin-A1 cells (red). The cyan line depict the point of collision (time; h: min).

(B) Distance ( m) traveled by the marginal cells 20 h after collision (mean ± SD).

(C) Time-lapse of the collision and subsequent cell division of a MDA-Cherry-ephrin-A1 cell (red) with a MDA-EGFP-EphA2 cell (green). Trans-illumination images (gray).

Capital letters indicates the donor cell (D), acceptor cell (A) and daughter cells (A', A'').

STAR Methods

LEAD CONTACT AND MATERIALS AVAILABILITY

Further information and request for reagents may be directed to, and will be fulfilled by the Lead Contact, Dr. Franck Perez (franck.perez@curie.fr). RUSH plasmids are already available through Addgene and we will deposit additional ones upon publication.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Cells and transfection

HeLa and MDA-MB231 wild-type cells were cultured in Dulbecco's modified Eagle medium (DMEM), high glucose, GlutaMAX™ (Life Technologies) supplemented with 10% fetal calf serum (FCS) (GE Healthcare), penicillin, streptomycin and 1mM sodium pyruvate (Life Technologies) at 37°C and 5% of CO2. HeLa and MDA-MB231 stable cell lines expressing the streptavidin-KDEL hook (Boncompain et al., 2012) and the SBP-Cherry-ephrin-A1 or SBP-EGFP-EphA2 reporters (cloned in a pCDH-CMV-MCS-EF1α-Puro vector) were generated by transduction with lentiviral particles generated in HEK293T cells and cultured as described above, but supplemented with 4 g/ml of puromycin (Invitrogen) and 0.5 mg/mL geneticin (Life Technologies). HeLa cells were transfected with calcium phosphate 24-48 h before fixation or imaging as previously described [START_REF] Jordan | Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation[END_REF].

METHOD DETAILS

Plasmids

RUSH plasmids of cargoes retain by their luminal domain (SBP-EGFP-ephrin-A1, SBP-Cherry-ephrin-A1, SBP-EGFP-ephrin-A5, SBP-EGFP-EphA2 ) were constructed as FseI/PacI inserts as previously described (Boncompain and Perez, 2012). SBP-Cherry-CD59 and SBP-Cherry-ALPP plasmids were previously published [START_REF] Fourriere | RAB6 and microtubules restrict protein secretion to focal adhesions[END_REF]. number of requests is high enough, those plasmids will be added to a public repository (e.g.

Addgene).

Immunofluorescence and antibodies

For immunofluorescence, cells were fixed for 15 min at room temperature with 3% paraformaldehyde (Electron Microscopy Sciences), washed in PBS and permeabilized with 0.05% saponin in PBS supplemented with 2 mg/mL of BSA. Coverslips were mounted in Mowiol (Calbiochem). Cell surface staining were performed with the primary antibody added in ice-cold PBS on non-fixed cells during 40 min as previously described (Boncompain and Perez, 2012). Cells were then fixed for 10 min with 2% paraformaldehyde. The mouse monoclonal anti-GFP antibody was purchased from Roche (Roche Cat# 11814460001, RRID:AB_390913; 1:300). The rabbit polyclonal Anti-Eph receptor A2+A3+A4 (phospho Y588 + Y596) antibody was purchased from abcam (Abcam Cat# ab62256, RRID:AB_942240; 1:500). The mouse monoclonal anti-LAMP1 antibody was purchased from Santa Cruz (Santa Cruz Biotechnology Cat# sc-20011, RRID:AB_626853; 1:500). The rabbit polyclonal anti-GFP (1:400), the mouse monoclonal Anti-phosphotyrosine (clone 4G10; 1:500) and the human monoclonal anti-giantin (clone TA10; 1:100) antibodies were obtained from the Recombinant Antibodies Platform (Institut Curie). Fluorescent-conjugated secondary antibodies were obtained from Jackson ImmunoResearch. SiR-DNA, SiR-Actin and SiR-Lysosome probes were purchased from Spirochrome, phalloidin-FITC was purchased from Sigma. Fixed cells were imaged with an epifluorescence microscope (Leica) equipped with a Coolsnap camera (Roper Scientific).

Live-cell imaging

Cells were seeded onto glass coverslips of 25 mm of diameter. Before imaging, coverslips were transferred into L-shape tubing Chamlide chamber (Live Cell Instrument). Chambers were filled with Leibovitz's medium (Life Technologies) pre-warmed at 37°C. For trafficking synchronization, the medium was removed and D-biotin (Sigma-Aldrich) at 40 µM in Leibovitz's medium was introduced using the tubing system of the chamber. Time-lapse imaging were obtained in a thermostat-controlled chamber at 37°C with an Eclipse 80i microscope (Nikon) equipped with a spinning disk confocal head (Perkin) and an Ultra897 iXon camera (Andor). Image acquisitions were performed using MetaMorph software (Molecular Device) on workstations of the PICT-IBiSA Lhomond Imaging facility of Institut Curie.

Treatments

Cells seeded on coverslips were pre-treated for 1 h at 37°C with either 0. Transferrin internalization was allowed by incubating the cells for 10 min at 37°C. Cells were then washed 3 times with DMEM-BSA and the remnant surface-bound Tfr-647 was stripped out by an acidic wash with Glycine buffer (0.1 M Glycine + 150 mM NaCl, pH 2.5, 30-40 sec). Finally, cells were washed in PBS, fixed with 3% PFA and mounted for imaging.

Degradative properties of the lysosomes were assessed by incubating the cells with DQ TM -Green-BSA (10 g/mL at 37°C) (ThermoFisher Scientific). Acidic lysosomes were visualized by treating cells with 75 nM LysoTracker Blue DND-22 (Invitrogen) at 37°C

and imaging them live for 5 min.

Arrest of the cell cycle at G2/M border was performed by treating the cells with 9 M RO3306 (Sigma-Aldrich) for 20 h at 37°C [START_REF] Vassilev | Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1[END_REF].

Cell confrontation assay

Cell confrontation assays were performed using cell culture inserts and 35 mm-High plates from Ibidi (Ibidi GmbH) following the instructions of the manufacturer as previously described [START_REF] Porazinski | EphA2 Drives the Segregation of Ras-Transformed Epithelial Cells from Normal Neighbors[END_REF]. Cell dyes were used for better visualization; MDA-SBP-Cherry-ephrin-A1 or MDA-SBP-EGFP-EphA2 cells were labeled in suspension with Cell Proliferation Dye eFluor® 670 or 5-(and 6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE-Cell Proliferation Dye) (Invitrogen), respectively (5 M, 10 min at 37°C). The labeling was stopped by incubating the cells with ice-cold DMEM medium supplemented with 20% FCS during 5 min, followed of three washes with 20% FCS medium (5 min, 1200

x g each). 3-4 x 10 4 cells were then seeded into each compartment of the insert and incubated in presence of 80 M of D-biotin at 37°C for 12-20 h before the removal of the insert. Upon reaching confluence, the insert was removed. Non-adhered cells were gently washed out with PBS and the dish was filled with 2 mL of 80 M D-biotin-containing DMEM. 8-12 h after insert removal the medium was switched to Leibovitz's medium supplemented with 80 M of D-biotin and 10% FBS. The cells were imaged-live using a spinning disk confocal microscope with a 20X objective.

Co-culture assay

HeLa cells were first electroporated with the appropriate plasmid and then mixed.

Electroporation were performed using the NEPA21 system (Nepa Gene Co.) according to the manufacturer's instructions. Briefly, cells were electroporated in Opti-MEM medium (Invitrogen) supplemented with 7 g/mL of avidin (Sigma-Aldrich) to neutralize the traces of biotin present in the medium. 1 x 10 6 of HeLa cells in suspension were mixed with 10 g of DNA up to a final volume of 100 L. For the electroporation of mRFP-Rab5-WT, mCherry-Rab5-Q79L, EGFP-Rab11-WT, DsRed-Rab11-S25N, EGFP-Rab7-WT and EGFP-Rab7-Q76L, 5 g of each plasmid were mixed with 5 g of an empty vector. Cells were then transferred to 2 mm gap cuvettes and electroporated using 2 poring pulses of 125 V, 7.5 msec length and 50 msec interval, followed by 5 transfer pulses of 20 V, 50 msec length and 50 msec interval. Equal amounts of donor and acceptor cells were then seeded onto 35 mm coverslip with pre-warmed medium and mixed by pipetting until a monodisperse cell suspension was achieved. Before imaging, cells were gently washed 3 times with PBS to remove dead cells and debris.

Ephrin inheritance assay

Wild-type MDA-MB231 cells (acceptor) were labeled in suspension with CFSE-Cell Proliferation Dye and seeded on gridded 35 mm-High plates from Ibidi (Ibidi GmbH)

following the instructions of the manufacturer. After 24h the cell cycle was arrested at G2/M transition using the reversible CDK1 inhibitor RO3306 (9 M, 20 h at 37°C) [START_REF] Vassilev | Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1[END_REF]. Then HeLa-SBP-Cherry-ephrin-A1 cells (donor) were seeded on top of the monolayer of acceptor cells and were let adhere in the presence of 40 M Dbiotin-containing DMEM for 2-4 h before imaging. The imaging field were chosen and the coordinates of the grid were registered. Then the cells were washed 5 times for 1 min with Leibovitz's medium and imaged live using a spinning disk confocal microscopy with a 63X objective as described before. After completion of mitosis, the cells were fixed for 15 min at room temperature with 3% paraformaldehyde and processed for immunofluorescence with antibodies against LAMP1 and pEphA receptors. The cells were located using the coordinates on the gridded plate and imaged using the same microscope and objective as above. Images were then manually aligned using Fiji software.

QUANTIFICATION AND STATISTICAL ANALYSIS

Image quantification

Normalized Golgi intensity was obtained by measuring the integrated intensity over the time in a mask of the Golgi apparatus, whose position was determined from latter time points as previously described (Boncompain et al., 2012). The same mask was then positioned in a cell-free region (background) and the integrated intensity over the time was

( D )

 D Intensity plots of SBP-EGFP-ephrin-A1 or EphA2-EGFP-SBP at the GA (mean ± SEM). (E) Percentage of colocalization (mean ± SEM) of EphA2-EGFP-SBP and SBP-Cherryephrin-A1 or SBP-EGFP-ephrin-A1 and SBP-Cherry-ephrin-A1. (D-E) Light red region is before Golgi exit, light green region is after Golgi exit. (F) EphA2-EGFP-SBP (top panel, green) or SBP-EGFP-ephrin-A1 (bottom panel, green) transfected cells 90 min after biotin addition. Nuclei (magenta), GA (red). Right panels (high-magnification images). Arrows indicate filopodia. (G) Live-cell imaging of the RUSH construct SBP-EGFP-ephrin-A1 (green) and the PM marker (PM-Cherry, red). Bottom panels (high-magnification images). Arrow indicates an ephrin-A1 cluster exported from filopodia. (H) Temporal projection of SBP-EGFP-ephrin-A1 and PM-Cherry imaged live. Bottom panels (high-magnification images). (I) Boxplot of the time of appearance of the first ephrin-cluster at the PM and the first ephrin-cluster exported from the PM. (C, F-H) Scale bars, 10 m. (*** t<0.001, two-tailed t-test) See also video S1 and S2.

Figure 2

 2 Figure 2 ephin-As are sorted into signaling filopodia

( D )

 D Live-cell imaging of SBP-EGFP-ephrin-A1 (green) and PM-Cherry (red) under control conditions (top panels) or cytochalasin-D treatment (bottom panels). F-actin (blue). (E) Kymographs analysis of one cluster of ephrin-A1 at cell-cell interface. The dotted line indicates the time of cluster formation. (F) Number of filopodia in control (DMSO) and cytochalasin-treated (Cyto-D) cells (mean ± SD). (G) Filopodia length distribution in control (DMSO) and cytochalasin-treated (Cyto-D) cells. (H) Boxplot showing the normalized number of transferred ephrin-A1 clusters in control (DMSO) and cytochalasin-D (Cyto-D) or latranculin A-treated (Lat-A) cells. (A-D) Scale bars, 10 m. (F, H) * t<0.05; ** t<0.01; ** t<0.01, two tailed t-test. See also video S3-S5.

Figure 3

 3 Figure 3 Cleavage-independent trans-endocytosis of ephrin-A1

  Same as in (D-F), but for the SBP-Cherry-ephrin-A1-TMD-ephrin-B1-EGFP sensor (drawn at the top). Red lines show a linear fit of the data. (A-B, D-E, G-H) Scale bars, 10 m. See also Fig. S1 and video S6.

Figure 4

 4 Figure 4 Dynamin-dependent trans-endocytosis of ephrin-A1 from cytonemes

Figure 5

 5 Figure 5 Localized trans-endocytosis of ephrin-A1 in complex with EphA2

Figure 6

 6 Figure 6 Long-lasting storage and signaling of trans-endocytosed ephrin-A1/EphA2 complexes in cancer cells

Figure 7

 7 Figure 7 Trans-endocytosed ephrin-A1 participates in contact-mediated repulsion

(

  D) Live-cell imaging of acceptor MDA-MB-231-WT cells labeled with a cell mask (A, green) co-cultured with donor MDA-Cherry-ephrin-A1 cells (D, red). Trans-illumination images (gray). The timeline at the top summarizes the different steps of the experiment. Time indicates the time after washout of RO3306 (top panels). Correlative immunostaining with antibodies against pEphARs (blue) and LAMP1 (magenta, bottom panels). Highmagnification of the boxed region (bottom right panels). Arrows indicate inherited ephrin-A1 clusters. (E) Percentage of trans-endocytosed ephrin-A1 clusters co-localizing with LAMP-1, pEphA or both (mean ± SEM). (F) Schematic model of the ephrin-As trans-endocytosis. (G) Functional overview. Removal of the Ephrin-A/EphA cell tether by trans-endocytosis permits cell-cell repulsion to occur. Long-distance communication through cytonemes may promote the sorting of EphARs to multivesicular bodies (MVB) and/or to signaling lysosomes. EphARs can be secreted in extracellular vesicles (EVs) interacting with Ephrin-As on distant cells (green cell). Ephrin-A-containing lysosomes may also be transferred to neighboring cells through TNTs. (A, C) Scale bars, 20 m; (D) scale bars, 10 m. (B, E) *t<0.05; ***t<0.001. two tailed ttest. See also video S10.

  5 M Cytochalasin D (Sigma-Aldrich), 0.5 M Latrunculin A (Interchim), 50 M TAPI-1 (Calbiochem), 20 M GI254023X (Tocris Bioscience), 2.5 Dasatinib (Cell Signaling Technology) or DMSO (Sigma-Aldrich) as a control. The trafficking of the reporters was synchronized by addition of biotin (40 µM at 37°C) in the presence of the respective drugs. For the Cytochalasin D and Latrunculin A treatments, we corroborated the disruption of the actin microfilaments by assessing the SiR-Actin staining (0.5 M, 1 h at 37°C). For TAPI-1 and GI254023X, the inhibition of ADAM metalloprotease was corroborated by using the SBP-Cherry-2XADAMtide-ephrin-A1-EGFP-GPI sensor. Inhibition of EphA2 signaling with Dasatinib was controlled by performing a pEphA immuno-staining. To assure that dynamin-dependent endocytosis was inhibited after Dyngo4A (Abcam) treatment, we checked for transferrin internalization. Briefly, cells seeded on coverslips were washed 3 times with PBS and starved from serum during 5 h at 37°C in DMEM supplemented with 0.1% BSA and penicillin/streptomycin (DMEM-BSA). Cells were then pre-treated with 10 M Dyngo4A or DMSO (Control) for 1h at 37°C in DMEM-BSA. The following incubation and washing steps were performed with DMEM-BSA containing either DMSO or 10 M Dyngo4A. The trafficking of SBP-EGFP-ephrin-A1 was synchronized by addition of biotin (40 µM, 90 min at 37°C). Cells were then incubated for 10 min on ice with 50 g/mL Transferrin Alexa Fluor TM 647 conjugate (Tfr-647, Molecular Probes) and washed 3 times with cold DMEM-BSA to remove the unbound Tfr-647.

  measured and subtracted to the integrated Golgi intensity. The values were normalized to the maximal value, and the measurements before the addition of biotin were adjusted to zero. Colocalization percentages over the time were determined in background subtracted cropped images containing individual cells. Single channel masks were generated by thresholding of each channel. The colocalization area was measured and normalized to the total area of each channel for every time point. The values were then translated in percentages. Random colocalization was determined by displacing the red channel 5x5 pixels in the xy plane for all the time points. The start of clustering and secretion were determined by visual inspection of videos of cells co-transfected with the RUSH reporter SBP-EGFP-ephrin-A1 and PM-Cherry to delimit the donor cell. The start of clustering was considered as the time point at which the first bright ephrin-A1 structure appeared at the PM of a given cell before to be exported. The start of the secretion was stated as the time point at which the first ephrin-A1 cluster was distinguishable separated from the PM of its donor cell. The normalized number of Merge images are shown (bottom row). Arrows indicate an example of a trans-endocytosed SBP-Cherry-ephrin-A1 cluster co-transported with SBP-EGFP-EphA2 towards a large lysosomal structure in an acceptor cell. (B) Single frame from live-cell imaging of MDA-MB231 cells stably expressing the RUSH construct SBP-Cherry-ephrin-A1 (SBP-Cherry-ephrin-A1, red) co-cultured with wild-type MDA-MB231 cells (left pannel). High magnification images of an acceptor cell are shown. Right panels shown the same cell after a short treatment with the acidotrophic dye Lysotracker (blue). Arrows indicate trans-endocytosed SBP-Cherry-ephrin-A1 in acidic lysosomes. (C) Live-cell imaging of MDA-MB231 cells stably expressing the RUSH construct SBP-Cherry-ephrin-A1 (SBP-Cherry-ephrin-A1, red) co-cultured with wild-type MDA-MB231 cells. Lysosomes were stained with the SiR-lysosome dye (blue). Degradative compartements were detected by the fluorogenic protease substrate DQ-BSA (green). Highmagnification images are presented below in black and white: SBP-Cherry-ephrin-A1 (top row), DQ-BSA (middle-upper row) and SiR-lysosome (middle-lower row). Merge images are shown (bottom row). Arrows indicate trans-endocytosed SBP-Cherry-ephrin-A1 in degradative lysosomes. Scale bars, 10 m.

Acknowledgments

The authors wish to thank V. Fraisier and L. Leconte from the PICT-IBiSA Lhomond Imaging facility of Institut Curie and the members of the Recombinant Antibodies Platform of the Institut Curie; Z. Gouveia for her help with lentiviral transductions; S. Divoux for her help with some experiments and G. Boncompain for constructs and advices; J. Ecard and M. Maurin for assistance with the Fiji macros. The work in the laboratory of F.P. was supported by the Institut Curie, the Centre National de la Recherche Scientifique (CNRS), the French Agence Nationale de la Recherche (ANR-12-BSV2-0003-01), Fondation Recherche Médicale (DEQ20120323723). LabEx CelTisPhyBio (ANR-10-LBX-0038 part of the IDEX PSL no. ANR-10-IDEX-0001-02; to FP and J.I.V.). Long-Term EMBO Fellowship (ALTF 607-2015) co-funded by the European Commission FP7 (Marie Curie Actions, LTFCOFUND2013, GA-2013-609409; to J.I.V.).

Author Contributions

J.I.V. conceived the study, conducted the experiments and analyzed the data. J.I.V. and F.P.

designed the experiments and wrote the manuscript. F.P. supervised the study and acquired funding.

Declaration of Interests

The authors declare no competing interests. (C) Time-lapse imaging of transport of SBP-Cherry-ephrin-A1 (red) and EphA2-EGFP-SBP (green) in a RUSH set-up (t = time after addition of biotin). Bottom panels (highmagnification images). transferred clusters was extracted from fixed epifluorescence images acquired with a 63X objective. The total number of exported clusters in each field was determined by manual counting and multiplied by the ratio of donor / acceptor cells.

For lysosomes-related quantifications, we measured the integrated intensity and area of hand-drawn region of interests (ROIs) comprising ephrin-A1-containing lysosomes, ephrin-A1-negative lysosomes or cytosolic lysosomes-free ROIs of the same size. For the cell confrontation assays we determined the distance traveled by the marginal cells after collision by measuring the distance between two parallels lines depicting the point of the first cell-to-cell contact and the position of the front of migration 20 h after this contact respectively. All these quantifications were made using Fiji software [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF] and Microsoft® Excel® 14.7.1 for drawing the plots. For boxplots; boxes represent 25 th and 75 th percentiles, the whiskers extend to the most extreme data points and the median is indicated as a band inside the box. The data were compared by using the twotailed t-test.

DATA AND CODE AVAILABILITY

This study did not generate/analyze datasets or codes

Additional information SUPPLEMENTARY INFORMATION

Correspondence and request for materials should be addressed to J.I.V. or F.P.

SUPPLEMENTARY VIDEOS

Supplementary Video 1 (Related to Figure 1