Multicolour imaging of post-Golgi sorting and trafficking in live cells.
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.
Keller
P
P
Cell Biology/Biophysics Programme, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
Toomre
D
D
Díaz
E
E
White
J
J
Simons
K
K
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D000818:Animals; D015415:Biomarkers; D002460:Cell Line; D016764:Cell Polarity; D003952:Diagnostic Imaging; D005089:Exocytosis / Q000502:physiology*; D005456:Fluorescent Dyes / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008561:Membrane Fusion / Q000502:physiology; D018613:Microscopy, Confocal; D021382:Protein Sorting Signals; D021381:Protein Transport / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D022161:Transport Vesicles / Q000378:metabolism*
D015415:Biomarkers; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins