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.

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Figure 1: Fusion of PGTIs with the basal membrane in MDCK cells.
Figure 2: Localization of Syntaxins 3 and 4 and Sec6 in MDCK cells.
Figure 3: Post-Golgi carrier movements are restricted within the apical cytoplasm.
Figure 4: Fusion of basolateral PGTIs with the lateral membrane in polarized MDCK cells.
Figure 5: Microtubule disruption selectively promotes fusion of apical PGTIs with the basal membrane.
Figure 6: Disruption of microtubules results in the selective depolarization of Syntaxin 3 in polarized MDCK cells.
Figure 7: Micro-injected Syntaxin 3 antibody inhibits fusion of apical PGTIs with the basal membrane in nocodazole-treated, polarized cells.

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Acknowledgements

This work was supported in part by a grant from the National Institutes of Health (GM34107) and by a Jules and Doris Stein professorship of the Research to Prevent Blindness Foundation (to E.R.-B.) and by an NIH National Research Service Award (EY06886, to G.K.). J.S. and S.M.S. acknowledge support from the National Science Foundation (BES0110070 and BES0119468, to S.M.S.). T.W. acknowledges support from the NIH (DK62338), the Department of Defense Prostate Cancer Research Program and the American Heart Association.

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Correspondence to Geri Kreitzer or Enrique Rodriguez-Boulan.

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Supplementary information

Supplementary Movie 1

Shows a single example of secretory carriers (containing p75-GFP) fusing with the basal membrane in non-polarized cells by TIR-FM. (AVI 5225 kb)

Supplementary Movie 2

Shows the spatial (Z-series) distributions of secretory carriers in polarized cells expressing p75-GFP or LDLR-GFP by confocal microscopy (AVI 4522 kb)

Supplementary Movie 3

Shows examples of fusing and non-fusing secretory carriers with the lateral membrane of polarized cells. There are two examples of fusing carriers and one example of a non-fusing carrier- I have piggy-backed the movies and inserted text frames to explain what the viewers are seeing. (AVI 992 kb)

Supplementary Movie 4

Shows the movie in which apical carriers can be induced to fuse with the basal membrane of polarized cells when treated with nocodazole. (AVI 3534 kb)

Supplementary Movie 5

Shows the fusion of a secretory carrier with the lateral membrane using multi-color imaging and our „color-based” fusion analysis. (AVI 1628 kb)

Supplementary Figures and Methods

Figure S1 Behaviors of basolateral PGTIs in polarized MDCK cells. (PDF 1328 kb)

Figure S2 Fusing GFP-containing PGTIs transiently become yellow when they fuse with DiIC16 labeled plasma membranes.

Figure S3 Recycling-defective LDLRa18-GFP does not colocalize with EEA- 1 in early endosomes after release of a Golgi block. up to 2hr) after release of the Golgi block. Cells were immunostained with EEA1

Supplementary Methods

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Kreitzer, G., Schmoranzer, J., Low, S. et al. Three-dimensional analysis of post-Golgi carrier exocytosis in epithelial cells. Nat Cell Biol 5, 126–136 (2003). https://doi.org/10.1038/ncb917

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