Essential cellular functions as diverse as genome maintenance and tissue morphogenesis rely on the dynamic organization of filamentous assemblies. For example, the precise structural organization of DNA filaments has profound consequences on all DNA-mediated processes including gene expression, whereas control over the precise spatial arrangement of cytoskeletal protein filaments is key for mechanical force generation driving animal tissue morphogenesis. Polarized fluorescence is currently used to extract structural organization of fluorescently labeled biological filaments by determining the orientation of fluorescent labels, however with a strong drawback: polarized fluorescence imaging is indeed spatially limited by optical diffraction, and is thus unable to discriminate between the intrinsic orientational mobility of the fluorophore labels and the real structural disorder of the labeled biomolecules. Here, we demonstrate that quantitative single-molecule polarized detection in biological filament assemblies allows not only to correct for the rotational flexibility of the label but also to image orientational order of filaments at the nanoscale using superresolution capabilities. The method is based on polarized direct stochastic optical reconstruction microscopy, using dedicated optical scheme and image analysis to determine both molecular localization and orientation with high precision. We apply this method to double-stranded DNA in vitro and microtubules and actin stress fibers in whole cells.iological processes are inherently driven by the molecularscale organization of biomolecular assemblies, which arrange in precise structures that are essential for biological functions in cells and tissues. The extent to which the biological function depends on the underlying molecular-scale organization is particularly evident in filamentous assemblies, such as DNA filaments and cytoskeletal protein filaments. Changes in the local higher-order organization of DNA filaments is tightly linked to essential DNA-mediated processes including control of gene expression, DNA replication, and DNA repair. However, how specific DNA-binding proteins affect DNA filament architecture and thus DNA-mediated functions is poorly understood (1). Similarly, the spatial organization of cytoskeletal filaments in cells and tissues is also weakly explored, despite their central role in generating forces and driving cell motility, cell division, and tissue morphogenesis (2). Electron microscopy has been widely used to provide molecular-scale images of the structure of such filament assemblies; however, it typically involves several daylong sample preparation and ultrathin sectioning of the biological material, thus limiting investigations in whole cells and tissues.Polarized fluorescence imaging is a powerful approach for elucidating the structural organization of filament assemblies because it is compatible with a wide variety of microscopy techniques, thus enabling studies across multiple spatial and temporal scales. Polarized fluorescenc...
While several clathrin-independent endocytic processes have been described so far, their biological relevance often remains elusive, especially in pathophysiological contexts such as cancer. In this study, we find that the tumor marker CD166/ALCAM (Activated Leukocyte Cell Adhesion Molecule) is a clathrin-independent cargo. We show that endophilin-A3-but neither A1 nor A2 isoforms-functionally associates with CD166-containing early endocytic carriers and physically interacts with the cargo. Our data further demonstrates that the three endophilin-A isoforms control the uptake of distinct subsets of cargoes. In addition, we provide strong evidence that the construction of endocytic sites from which CD166 is taken up in an endophilin-A3-dependent manner is driven by extracellular galectin-8. Taken together, our data reveal the existence of a previously uncharacterized clathrin-independent endocytic modality, that modulates the abundance of CD166 at the cell surface, and regulates adhesive and migratory properties of cancer cells.
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