Abstract
Although epithelial cells are known to exhibit a polarized distribution of membrane components, the pathways responsible for delivering membrane proteins to their appropriate domains remain unclear. Using an optimized approach to three-dimensional live cell imaging, we have visualized the transport of newly synthesized apical and basolateral membrane proteins in fully polarized filter-grown Madin-Darby canine kidney cells. We performed a detailed quantitative kinetic analysis of trans-Golgi network (TGN) exit, passage through transport intermediates, and arrival at the plasma membrane using cyan/yellow fluorescent protein-tagged glycosylphosphatidylinositol-anchored protein and vesicular stomatitis virus glycoprotein as apical and basolateral reporters, respectively. For both pathways, exit from the TGN was rate limiting. Furthermore, apical and basolateral proteins were targeted directly to their respective membranes, resolving current confusion as to whether sorting occurs on the secretory pathway or only after endocytosis. However, a transcytotic protein did reach the apical surface after a prior appearance basolaterally. Finally, newly synthesized proteins appeared to be delivered to the entire lateral or apical surface, suggesting-contrary to expectations-that there is not a restricted site for vesicle docking or fusion adjacent to the junctional complex.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism
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Cell Line
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Cell Membrane / metabolism
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Cell Polarity / physiology*
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Dogs
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Endocytosis / physiology
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Epithelial Cells / cytology
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Epithelial Cells / metabolism*
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Glycoproteins / genetics
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Glycoproteins / metabolism
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Glycosylphosphatidylinositols / metabolism
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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Kinetics
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Laser Scanning Cytometry / methods*
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Luminescent Proteins / genetics
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Luminescent Proteins / metabolism
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Membrane Glycoproteins / genetics
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Membrane Glycoproteins / metabolism
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Membrane Proteins / genetics
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Membrane Proteins / metabolism*
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Neural Cell Adhesion Molecules / genetics
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Neural Cell Adhesion Molecules / metabolism
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Protein Transport / physiology
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Recombinant Fusion Proteins / metabolism
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Temperature
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Transfection
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Transport Vesicles / metabolism
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Viral Envelope Proteins / genetics
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Viral Envelope Proteins / metabolism
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trans-Golgi Network / metabolism
Substances
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Bacterial Proteins
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Cyan Fluorescent Protein
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G protein, vesicular stomatitis virus
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Glycoproteins
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Glycosylphosphatidylinositols
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Luminescent Proteins
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Membrane Glycoproteins
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Membrane Proteins
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Neural Cell Adhesion Molecules
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Recombinant Fusion Proteins
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Viral Envelope Proteins
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yellow fluorescent protein, Bacteria
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Green Fluorescent Proteins