Three-dimensional analysis of post-Golgi carrier exocytosis in epithelial cells

Abstract

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.

Unravelling protein sorting

“…2c). This confirms the existence of a direct apical route from the TGN to the apical membrane domain, as previously demonstrated by quantitative imaging of post-Golgi vesicle fusion 5 and by abundant biochemical data (reviewed in ref. 1).…”

“…Thus, these post-TGN carriers were destined for the basolateral surface, and not the apical membrane. These carriers accumulated immediately adjacent to the tight junction, a sub-domain of the lateral membrane shown previously to be the site for vesicle delivery 5 and to contain the Sec6/8-exocyst protein complex 6 . Incubation of the apical membrane domain with tannic acid did not perturb delivery of VSVG-tsO45-CFP to the basolateral membrane (Fig.…”

“…Clearly, sorting of proteins occurs in the TGN, but the general nature of this process in different epithelial cells needs to be re-evaluated as powerful imaging techniques and procedures to interfere with specific steps in the pathway become available. Another issue is the precise site of fusion: the authors found that delivery of carriers containing GFP-GPI was restricted to the apex of the lateral membrane, a region previously shown to contain the Sec6/8-exocyst complex and to be a site for docking and fusion of basolateral vesicles with the plasma membrane 5,6 . It remains unknown whether vesicle delivery -for example, of p75 -to the apical membrane also occurs at this site.…”

The epithelial polarity program: machineries involved and their hijacking by cancer

“…Live imaging experiments using model apical or basolateral PM proteins tagged with GFP (Kreitzer et al, 2003) demonstrated that non polarized subconfluent MDCK cells fuse apical and basolateral transport vesicles randomly with the PM, due to the non-polarized distributions of syntaxins 3 and 4. Polarized MDCK cells, in contrast, dramatically reorganize their delivery routes from the Golgi and RE, which are localized supranuclearly in these cells due to repositioning of the MTOC, to the PM.…”

“…Polarized MDCK cells, in contrast, dramatically reorganize their delivery routes from the Golgi and RE, which are localized supranuclearly in these cells due to repositioning of the MTOC, to the PM. Apical transport carriers fuse with the apical PM domain, promoted by the apical localization of syntaxin 3, whereas basolateral transport carriers fuse with a 'hot spot' in the junctional region of the lateral membrane, guided by the restricted presence of syntaxin 4 at the lateral membrane (Kreitzer et al, 2003) and the enrichment of the tethering particle exocyst at the junctional region (Grindstaff et al, 1998).…”