21 February 2017 Correlated fluorescence-atomic force microscopy studies of the clathrin mediated endocytosis in SKMEL cells
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Clathrin-mediated endocytosis (CME) is one of the central pathways for cargo transport into cells, and plays a major role in the maintenance of cellular functions, such as intercellular signaling, nutrient intake, and turnover of plasma membrane in cells. The clathrin-mediated endocytosis process involves invagination and formation of clathrin-coated vesicles. However, the biophysical mechanisms of vesicle formation are still debated. Currently, there are two models describing membrane bending during the formation of clathrin cages: the first involves the deposition of all clathrin molecules to the plasma membrane, forming a flat lattice prior to membrane bending, whereas in the second model, membrane bending happens simultaneously as the clathrin arrives to the site to form a clathrin-coated cage. We investigate clathrin vesicle formation mechanisms through the utilization of tapping-mode atomic force microscopy for high resolution topographical imaging in neutral buffer solution of unroofed cells exposing the inner membrane, combined with fluorescence imaging to definitively label intracellular constituents with specific fluorophores (actin filaments labeled with green phalloidin and clathrin coated vesicles with the fusion protein Tq2) in SKMEL (Human Melanoma) cells. An extensive statistical survey of many hundreds of CME events, at various stages of progression, are observed via this method, allowing inferences about the dominant mechanisms active in CME in SKMEL cells. Results indicate a mixed model incorporating aspects of both the aforementioned mechanisms for CME.
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Amy Hor, Amy Hor, Anh Luu, Anh Luu, Lin Kang, Lin Kang, Brandon Scott, Brandon Scott, Elizabeth Bailey, Elizabeth Bailey, Adam Hoppe, Adam Hoppe, Steve Smith, Steve Smith, } "Correlated fluorescence-atomic force microscopy studies of the clathrin mediated endocytosis in SKMEL cells", Proc. SPIE 10071, Single Molecule Spectroscopy and Superresolution Imaging X, 100710I (21 February 2017); doi: 10.1117/12.2256683; https://doi.org/10.1117/12.2256683

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