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...

The Peroxiredoxin 1 (PRDX1) gene maps to chromosome arm 1p and is hemizygously deleted and epigenetically silenced in isocitrate dehydrogenase 1 or 2 (IDH)-mutant and 1p/19q-codeleted oligodendroglial tumors. In contrast, IDH-wildtype astrocytic gliomas including glioblastomas mostly lack epigenetic silencing and express PRDX1 protein. In our study, we investigated how PRDX1 contributes to the infiltrative growth of IDH-wildtype gliomas. Focusing on p38α-dependent pathways, we analyzed clinical data from 133 patients of the NOA-04 trial cohort to look for differences in the gene expression profiles of gliomas with wildtype or mutant IDH. Biochemical interaction studies as well as in vitro and ex vivo migration studies were used to establish a biological role of PRDX1 in maintaining pathway activity. Whole-brain high-resolution ultramicroscopy and survival analyses of pre-clinical mouse models for IDH-wildtype gliomas were then used for in vivo confirmation. Based on clinical data, we found that the absence of PRDX1 is associated with changes in the expression of MET/HGF signaling components. PRDX1 forms a heterodimer with p38α mitogen-activated protein kinase 14 (MAPK14), stabilizing phospho-p38α in glioma cells. This process amplifies hepatocyte growth factor (HGF)-mediated signaling and stimulates actin cytoskeleton dynamics that promote glioma cell migration. Whole-brain high-resolution ultramicroscopy confirms these findings, indicating that PRDX1 promotes glioma brain invasion in vivo. Finally, reduced expression of PRDX1 increased survival in mouse glioma models. Thus, our preclinical findings suggest that PRDX1 expression levels may serve as a molecular marker for patients who could benefit from targeted inhibition of MET/HGF signaling.

Clathrin-mediated endocytosis is one of the major pathways by which cells internalise cargo molecules. Recently it has been shown that clathrin triskelia can first assemble as flat lattices before the membrane starts to bend. However, for fully assembled clathrin lattices high energetic and topological barriers exist for the flat-to-curved transition. Here we explore the possibility that flat clathrin lattices grow with vacancies that are not visible in traditional imaging techniques but would lower these barriers. We identify the Eden model for cluster growth as the most appropriate modeling framework and systematically derive the four possible variants that result from the specific architecture of the clathrin triskelion. Our computer simulations show that the different models lead to clear differences in the statistical distributions of cluster shapes and densities. Experimental results from electron microscopy and correlative light microscopy provide first indications for the model variants with a moderate level of lattice vacancies.

One of the most fundamental processes of the cell is the uptake of molecules from the surrounding environment. Clathrin-mediated endocytosis (CME) is the best-described uptake pathway and regulates nutrient uptake, protein and lipid turnover at the plasma membrane (PM), cell signaling, cell motility and cell polarity. The main protein in CME is clathrin, which assembles as a triskelion-looking building block made of three clathrin heavy chains and three clathrin light chains. Compared to clathrin heavy chains (CHCs), the role of the two isoforms of clathrin light chains (CLCA and CLCB) is poorly understood. Here, we confirm that the simultaneous deletion of both CLCA/B causes abnormal actin structures at the ventral PM and we describe them, for the first time, as functional invadopodia rather than disorganized actin-cytoskeleton assembly sites. Their identification is based on the occurrence of common invadopodia markers as well as functional invadopodia activity characterized by an increased local proteolytic activity of the extracellular matrix proteins. We demonstrate that CLCA/B deletion impacts the intracellular trafficking and recovery of the matrix metalloproteinase 14 (MMP14) leading to its accumulation at the plasma membrane and induction of invadopodia formation. Importantly, we show that invadopodia formation can be prevented by depletion of MMP14. As such, we propose that CLCA/B regulate invadopodia formation by regulating MMP14 delivery to the plasma membrane.

Although essential for many cellular processes, the sequence of structural and molecular events during clathrin-mediated endocytosis remains elusive. While it was believed that clathrin-coated pits grow with a constant curvature, it was recently suggested that clathrin first assembles to form a flat structure and then bends while maintaining a constant surface area. Here, we combine correlative electron and light microscopy and mathematical modelling to quantify the sequence of ultrastructural rearrangements of the clathrin coat during endocytosis in mammalian cells. We confirm that clathrin-coated structures can initially grow flat and that lattice curvature does not show a direct correlation with clathrin coat assembly. We demonstrate that curvature begins when 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 model that mammalian cells dynamically regulate the flat-to-curved transition in clathrin-mediated endocytosis by both biochemical and mechanical factors.