Abstract
The biogenesis and maintenance of asymmetry is crucial to many cellular functions including absorption and secretion, signalling, development and morphogenesis. Here we have directly visualized the segregation and trafficking of apical (glycosyl phosphatidyl inositol-anchored) and basolateral (vesicular stomatitis virus glycoprotein) cargo in living cells using multicolour imaging of green fluorescent protein variants. Apical and basolateral cargo segregate progressively into large domains in Golgi/trans-Golgi network structures, exclude resident proteins, and exit in separate transport containers. These remain distinct and do not merge with endocytic structures suggesting that lateral segregation in the trans-Golgi network is the primary sorting event. Fusion with the plasma membrane was detected by total internal reflection microscopy and reveals differences between apical and basolateral carriers as well as new 'hot spots' for exocytosis.
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Acknowledgements
We acknowledge Olympus and TILL Photonics for providing equipment and support to the Advanced Light Microscope Facility at EMBL; J. Rietdorf and R. Pepperkok for technical support; R. Tsien and G. Banting for ECFP and TGN38–EGFP cDNA, respectively; and N. LeBot and J. Füllekrug for GFP and gp27 antibodies, respectively. J. Ellenberg, G. Griffiths and R. Pepperkok are acknowledged for critical comments on the manuscript. P.K. was supported by a grant from the Max Plank Gesellschaft, D.T. by a Marie Curie Research grant, E.D. by a grant from the Secretaría de Estado de Educación, Universidades, Investigación y Desarollo, and K.S. by an EU network grant and a grant from the Deutsche Forschungsgemeinschaft. Supplementary Information is available on http://www.mpi-cbg.de/content.php3?lang=en&aktID=videos8.
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Keller, P., Toomre, D., Díaz, E. et al. Multicolour imaging of post-Golgi sorting and trafficking in live cells . Nat Cell Biol 3, 140–149 (2001). https://doi.org/10.1038/35055042
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DOI: https://doi.org/10.1038/35055042
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