<> "The repository administrator has not yet configured an RDF license."^^ . <> . . "A nanoscopic reconstruction of clathrin coat remodeling during mammalian endocytosis"^^ . "Clathrin-mediated endocytosis is an essential pathway by which eukaryotic cells take up extracellular compounds. It is characterized by the formation of a clathrin coat at the inner leafet of the plasma membrane, which drives the invagination of the membrane to form a spherical vesicle.\r\n\r\nWhile the static ultrastructure and the interactome of the clathrin coat are well-studied, the molecular mechanisms and dynamics of the structural rearrangements that reshape the underlying membrane remain controversial. Convergence toward a detailed mechanistic understanding has been difficult due to the small size, molecular complexity, and relatively fast dynamics of the endocytic process.\r\n\r\nIn this work, I present a superresolution microscopy approach that allowed me to dynamically reconstruct the 3D architecture of clathrin coats in situ. To robustly determine the geometry of these structures, I employed the model-fitting framework LocMoFit, which approximates each clathrin coat as a spherical cap. The parameters extracted with this approach accurately describe geometric quantities such as coat curvature and surface area and enable the sorting of structures along their endocytic progression. The reconstruction of changes in clathrin coat geometry showed that these structures initially pre-assemble on a flat membrane, initiating curvature once approximately half the final coat area has been accumulated. Curvature then increases while clathrin polymerization continues until the final vesicular coat is formed. Based on these findings, we formulated the cooperative curvature model, which is based on positive feedback for curvature generation. This model accurately describes the experimental data retrieved from three cell lines, thus likely conceptualizing a general, physiologically relevant pathway of membrane bending by the clathrin coat.\r\n\r\nGeometric changes in the clathrin coat are a result of the cumulative contributions of over 50 endocytic components involved in this process. Concurrently visualizing multiple components of the endocytic machinery could elucidate the detailed interplay of the involved proteins. As proof of principle, I visualized the dynamic recruitment of dynamin-2 and adaptor protein-2 complex relative to the developing clathrin coat using dual-color imaging. Based on the spatiotemporal distribution of these proteins, their functional role within the larger endocytic machinery could be confirmed.\r\n\r\nTaken together, the superresolution microscopy approach I present in this work allows the reliable dynamic reconstruction of clathrin coat remodeling at the nanoscale. The resulting observations informed a new clathrin coat growth model, allowing for further mechanistic insights into the process. Future endeavors will focus on the application of this approach to reconstruct the temporal rearrangement of other components to the endocytic site, progressing towards a structural model of the entire endocytic machinery at the nanoscale. This pipeline further offers a means to test for structural adaptations of the endocytic machinery in response to changing environmental conditions."^^ . "2023" . . . . . . . "Aline"^^ . "Tschanz"^^ . "Aline Tschanz"^^ . . . . . . "A nanoscopic reconstruction of clathrin coat remodeling during mammalian endocytosis (PDF)"^^ . . . "Thesis_AlineTschanz_V3.pdf"^^ . . . "A nanoscopic reconstruction of clathrin coat remodeling during mammalian endocytosis (Other)"^^ . . . . . . "indexcodes.txt"^^ . . . "A nanoscopic reconstruction of clathrin coat remodeling during mammalian endocytosis (Other)"^^ . . . . . . "lightbox.jpg"^^ . . . "A nanoscopic reconstruction of clathrin coat remodeling during mammalian endocytosis (Other)"^^ . . . . . . "preview.jpg"^^ . . . "A nanoscopic reconstruction of clathrin coat remodeling during mammalian endocytosis (Other)"^^ . . . . . . "medium.jpg"^^ . . . "A nanoscopic reconstruction of clathrin coat remodeling during mammalian endocytosis (Other)"^^ . . . . . . "small.jpg"^^ . . "HTML Summary of #33745 \n\nA nanoscopic reconstruction of clathrin coat remodeling during mammalian endocytosis\n\n" . "text/html" . . . "570 Biowissenschaften, Biologie"@de . "570 Life sciences"@en . .