Although essential for many cellular processes, the sequence of structural and molecular events during clathrin-mediated endocytosis remains elusive. While it was long believed that clathrin-coated pits grow with a constant curvature, it was recently suggested that clathrin first assembles to form flat structures that then bend while maintaining a constant surface area. Here, we combine correlative electron and light microscopy and mathematical growth laws to study the ultrastructural rearrangements of the clathrin coat during endocytosis in BSC-1 mammalian cells. We confirm that clathrin coats initially grow flat and demonstrate that curvature begins when around 70% of the final clathrin content is acquired. We find that this transition is marked by a change in the clathrin to clathrin-adaptor protein AP2 ratio and that membrane tension suppresses this transition. Our results support the notion that BSC-1 mammalian cells dynamically regulate the flat-to-curved transition in clathrin-mediated endocytosis by both biochemical and mechanical factors.

The homeostatic chemokines CCL19 and CCL21 and their common cognate chemokine receptor CCR7 orchestrate immune cell trafficking by eliciting distinct signaling pathways. Here, we demonstrate that human CCR7 is N-glycosylated on 2 specific residues in the N terminus and the third extracellular loop. Conceptually, CCR7 glycosylation adds steric hindrance to the receptor N terminus and extracellular loop 3, acting as a "swinging door" to regulate receptor sensitivity and cell migration. We found that freshly isolated human B cells, as well as expanded T cells, but not naïve T cells, express highly sialylated CCR7. Moreover, we identified that human dendritic cells imprint T cell migration toward CCR7 ligands by secreting enzymes that deglycosylate CCR7, thereby boosting CCR7 signaling on T cells, permitting enhanced T cell locomotion, while simultaneously decreasing receptor endocytosis. In addition, dendritic cells proteolytically convert immobilized CCL21 to a soluble form that is more potent in triggering chemotactic movement and does not desensitize the receptor. Furthermore, we demonstrate that soluble CCL21 functionally resembles neither the CCL19 nor the CCL21 phenotype but acts as a chemokine with unique features. Thus, we advance the concept of dendritic cell-dependent generation of micromilieus and lymph node conditioning by demonstrating a novel layer of CCR7 regulation through CCR7 sialylation. In summary, we demonstrate that leukocyte subsets express distinct patterns of CCR7 sialylation that contribute to receptor signaling and fine-tuning chemotactic responses.

Intestinal epithelial cells (IECs) constitute the primary barrier that separates us from the outside environment. These cells, lining the surface of the intestinal tract, represent a major challenge that enteric pathogens have to face. How IECs respond to viral infection and whether enteric viruses have developed strategies to subvert IECs innate immune response remains poorly characterized. Using mammalian reovirus (MRV) as a model enteric virus, we found that the intermediate subviral particles (ISVPs), which are formed in the gut during the natural course of infection by proteolytic digestion of the reovirus virion, trigger reduced innate antiviral immune response in IECs. On the contrary, infection of IECs by virions induces a strong antiviral immune response that leads to cellular death. Additionally, we determined that virions can be sensed by both TLR and RLR pathways while ISVPs are sensed by RLR pathways only. Interestingly, we found that ISVP infected cells secrete TGF-β acting as a pro-survival factor that protects IECs against virion induced cellular death. We propose that ISVPs represent a reovirus strategy to initiate primary infection of the gut by subverting IECs innate immune system and by counteracting cellular-death pathways.

18Clathrin is a unique scaffold protein, which forms polyhedral lattices with flat and curved 19 morphology. The function of curved clathrin-coated pits in forming endocytic structures is 20 well studied. On the contrary, the role of large flat clathrin arrays, called clathrin-coated 21 plaques, remains ambiguous. Previous studies suggested an involvement of plaques in cell 22 adhesion. However, the molecular origin leading to their formation and their precise 23 functions remain to be determined. Here, we study the origin and function of clathrin-24 coated plaques during cell migration. We revealed that plaque formation is intimately 25 linked to extracellular matrix (ECM) modification by focal adhesions (FAs). We show that in 26 migrating cells, FAs digest the ECM creating extracellular topographical cues that dictate the 27 future location of clathrin-coated plaques. We identify Eps15 and Eps15R as key regulators 28 for the formation of clathrin-coated plaques at locally remodelled ECM sites. Using a genetic 29 silencing approach to abrogate plaque formation and 3D-micropatterns to spatially control 30 the location of clathrin-coated plaques, we could directly correlate cell migration 31 directionality with the formation of clathrin-coated plaques and their ability to recognize 32 extracellular topographical cues. We here define the molecular mechanism regulating the 33 functional interplay between FAs and plaques and propose that clathrin-coated plaques act 34 as regulators of cell migration promoting contact guidance-mediated collective migration in 35 a cell-to-cell contact independent manner. 36. 37 the plasma membrane to form a highly dynamic array 22,23 . During clathrin-mediated 64 endocytosis (CME), small transient clathrin coats can form initially as curved or flat arrays 65 which will rearrange to form clathrin-coated pits (CCPs) with a typical diameter of 100-150 66 nm [24][25][26] . Distinct from these small endocytic structures, flat long-lived larger clathrin coats, 67 known as clathrin-coated plaques, are frequently observed 27 . These clathrin-coated plaques 68 appear to have pleiotropic functions. They can facilitate endocytosis and signalling via 69 clustering of plasma membrane receptors and nucleating endocytic events [28][29][30] . The putative 70 role of larger clathrin arrays in cell adhesion and migration has long been discussed 31,32 . It 71 has recently been readdressed with the observation that specialized clathrin arrays named 72 tubular clathrin/AP2 lattices (TCALs) are responsible for binding collagen fibres in a 3D-73 environment 19 and that association of clathrin-coated plaques with the ECM is integrin 74 dependent 27,33,34 . 75 To date, the mechanisms that lead to clathrin-coated plaque formation and stabilization at 76 the plasma membrane and the cellular and extracellular determinants that dictate whether 77 a clathrin-coated plaque displays an endocytic or a non-endocytic function are still unclear. 78Similarly, the molecular and/or physical determinants that drive cl...