Three-dimensional analysis of post-Golgi carrier exocytosis in epithelial cells.
Targeted delivery of proteins to distinct plasma membrane domains is critical to the development and maintenance of polarity in epithelial cells. We used confocal and time-lapse total internal reflection fluorescence microscopy (TIR-FM) to study changes in localization and exocytic sites of post-Golgi transport intermediates (PGTIs) carrying GFP-tagged apical or basolateral membrane proteins during epithelial polarization. In non-polarized Madin Darby Canine Kidney (MDCK) cells, apical and basolateral PGTIs were present throughout the cytoplasm and were observed to fuse with the basal domain of the plasma membrane. During polarization, apical and basolateral PGTIs were restricted to different regions of the cytoplasm and their fusion with the basal membrane was completely abrogated. Quantitative analysis suggested that basolateral, but not apical, PGTIs fused with the lateral membrane in polarized cells, correlating with the restricted localization of Syntaxins 4 and 3 to lateral and apical membrane domains, respectively. Microtubule disruption induced Syntaxin 3 depolarization and fusion of apical PGTIs with the basal membrane, but affected neither the lateral localization of Syntaxin 4 or Sec6, nor promoted fusion of basolateral PGTIs with the basal membrane.
Kreitzer
Geri
G
Margaret M. Dyson Vision Research Institute, Weill Medical College of Cornell University, New York, NY 10021, USA. ggurlan@med.cornell.edu
Schmoranzer
Jan
J
Low
Seng Hui
SH
Li
Xin
X
Gan
Yunbo
Y
Weimbs
Thomas
T
Simon
Sanford M
SM
Rodriguez-Boulan
Enrique
E
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D016764:Cell Polarity; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005089:Exocytosis / Q000502:physiology*; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008561:Membrane Fusion / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism; D008845:Microinjections; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism; D015739:Nocodazole / Q000494:pharmacology; D021381:Protein Transport; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors
D000906:Antibodies; D000970:Antineoplastic Agents; D002352:Carrier Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D015739:Nocodazole