In this regard, it will be important to determine in the future whether receptors other than integrins may exploit trafficking to the TGN to convey signals modulating the biosynthetic routes according to the presence, amount and location of extracellular cues in polarized cells (Supplementary Fig. sites. ncomms13546-s3.avi (758K) GUID:?18FF6C65-EF6C-47FA-8551-74B3215170C9 Supplementary Movie 3 Live time-lapse confocal fluorescence microscopy of MO-injected zebrafish embryo at 48 AG14361 hpf. Time-lapse confocal fluorescence microscopy reveals normal heart beating in a representative 48 hpf MO-injected zebrafish embryo. ncomms13546-s4.avi (408K) GUID:?098A6D2F-41AB-4049-8D1D-DAF5CEDD1C44 Supplementary Movie 4 Live time-lapse phase contrast microscopy of MO-injected zebrafish embryo at 48 hpf. Time-lapse phase contrast microscopy confirms normal heart beating in the same 48 hpf MO-injected zebrafish embryo displayed in movie S3. ncomms13546-s5.avi (581K) GUID:?7F9246E7-735D-4C0A-9B3E-F181B781B211 Data Availability StatementThe authors declare that all data supporting the findings of this study are available within the article and its Supplementary Information files or are available from the corresponding author upon request. Abstract Basolateral polymerization of cellular fibronectin (FN) into a meshwork drives endothelial cell (EC) polarity and vascular remodelling. However, mechanisms coordinating 51 integrin-mediated extracellular FN endocytosis AG14361 and exocytosis of newly synthesized FN remain elusive. Here we show that, on Rab21-elicited internalization, FN-bound/active 51 is recycled to the EC surface. We identify a pathway, comprising the AG14361 regulators of post-Golgi carrier formation PI4KB and AP-1A, the small GTPase Rab11B, the surface tyrosine phosphatase receptor PTPRF and its adaptor PPFIA1, which we propose acts as a funnel combining FN secretion and recycling of active 51 integrin from the and in the developing zebrafish embryo. Essentially all morphogenetic events of multicellular organisms, including angiogenesis1, require a highly dynamic adhesion to extracellular matrix (ECM) proteins that is mediated primarily by integrins2. During vascular development, the interaction between fibronectin (FN) and its major receptor 51 integrin induces apico-basal polarity of endothelial cells (ECs) and the formation of the single lumen of blood vessels3,4. Integrin-mediated ECM signalling controls the spatial orientation of the apico-basal axis not only in ECs5, but also in several other polarized cell types6. Cells secrete FN as a soluble dimer that is assembled by integrins into a fibrillar network. The latter represents the bioactive form of FN and is capable of inducing mechanical and chemical signals that are essential for EC tubulogenesis7 and angiogenesis1,8. Although the fibrillar FN network is cross-linked and associated with other ECM proteins8, it is continuously turned over and remodelled due to matrix metalloproteinase-dependent cleavage9 and integrin-mediated endocytosis of freed FN proteolytic fragments10,11,12,13. Indeed, a complex machinery composed of the transmembrane glycoprotein neuropilin 1 (Nrp1), its cytosolic interactor GAIP interacting protein C terminus, member 1 (GIPC1) and the associated motor myosin VI (MYO6) selectively controls the endocytosis of FN fragments bound to active 51 integrins from the surface of ECs11,14. The importance of this process is highlighted by the severe impairment of FN fibrillogenesis that occurs in ECs on disruption of Nrp1 signaling14. Since internalized active 51 integrins recycle back to the cell surface15,16, it has been proposed that they may also accomplish other tasks in addition to the endocytosis of FN AG14361 fragments, such as the binding of newly synthesized FN in the exocytosis pathway, resulting in its deposition and polymerization at the basolateral surface of ECs to replenish degraded FN fibrils9,10,14. RTP801 Despite the attractiveness of this hypothesis and the fact that the turnover of the fibrillar FN network is essential for physiologic as AG14361 well as pathologic angiogenesis, it is unknown where in the cell 51 integrins may be loaded with newly synthesized FN molecules and how the exocytosis of the FN-51 integrin complex could be orchestrated at the molecular level. In polarized cells, the asymmetric distribution of transmembrane and secreted soluble proteins, for example, the selective basolateral deposition of FN fibrils in ECs17,18,19, relies on the unique sorting properties of the TGN along the biosynthetic routes20,21. Specific apical and basal endosomal compartments are thought to control the local recycling of endocytosed receptors22. However, recent studies suggest that internalized integrins may also reach the TGN before being recycled back to the plasma membrane23,24,25,26. Liprins.