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Clathrin is a key regulator of basolateral polarity

Nature volume 452, pages 719–723 (2008)Cite this article

Abstract

Clathrin-coated vesicles are vehicles for intracellular trafficking in all nucleated cells, from yeasts to humans. Many studies have demonstrated their essential roles in endocytosis and cellular signalling processes at the plasma membrane. By contrast, very few of their non-endocytic trafficking roles are known, the best characterized being the transport of hydrolases from the Golgi complex to the lysosome. Here we show that clathrin is required for polarity of the basolateral plasma membrane proteins in the epithelial cell line MDCK. Clathrin knockdown depolarized most basolateral proteins, by interfering with their biosynthetic delivery and recycling, but did not affect the polarity of apical proteins. Quantitative live imaging showed that chronic and acute clathrin knockdown selectively slowed down the exit of basolateral proteins from the Golgi complex, and promoted their mis-sorting into apical carrier vesicles. Our results demonstrate a broad requirement for clathrin in basolateral protein trafficking in epithelial cells.

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Figure 1: Clathrin knockdown in MDCK cells.
Figure 2: Clathrin-depleted cells develop normal tight junctions.
Figure 3: Clathrin knockdown depolarizes basolateral proteins at steady state.
Figure 4: Clathrin knockdown depolarizes surface delivery of basolateral proteins.
Figure 5: Clathrin suppression disrupts exit and sorting of basolateral proteins at the TGN.

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Acknowledgements

We thank A. Müsch, T. McGraw and T. Ryan for comments on the manuscript. This work received support from NIH grants to E.R.-B., from the Dyson Foundation and from the Research to Prevent Blindness Foundation. E.P. was supported by a fellowship from Association pour la Recherche sur le Cancer during the initial stages of this work. R.S. was supported by a training predoctoral grant from the NIH.

Author Contributions S.D. designed research; S.D., E.P., D.G., R.S., S.B.S. and A.D. performed research; S.D. and E.R.-B. analysed data and wrote the paper.

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Author notes

  1. Emilie Perret and Diego Gravotta: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Ophthalmology, Dyson Vision Research Institute, LC-300, and,

    Sylvie Deborde, Emilie Perret, Diego Gravotta, Ami Deora, Susana Salvarezza, Ryan Schreiner & Enrique Rodriguez-Boulan

  2. Department of Cell and Developmental Biology, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10065, USA,

    Enrique Rodriguez-Boulan

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Corresponding author

Correspondence to Enrique Rodriguez-Boulan.

Supplementary information

Supplementary information

The file contains Supplementary Figures 1-4 with Legends. (PDF 746 kb)

The file contains Supplementary Movie 1.

This movie shows the exit from Golgi of NCAM-GFP in cells expressing HA-FKBP-LC treated or not with crosslinker AP20187. The time lapse movie covers a period of 2h, during which time NCAM leaves the perinuclear region and reaches the plasma membrane. It is evident that the exit from Golgi is delayed when clathrin function is impaired. Bar = 20 m. (MOV 1348 kb)

The file contains Supplementary Movie 2.

This movie shows the exit from Golgi of both NCAM-cherry and p75-GFP in a control MDCK cell starting 10-15 min after release of the 20oC Golgi block. Boxes indicate areas zoomed below to higher magnification to allow visualization of representative post-Golgi carriers containing either p75-GFP or NCAM-cherry, but not both. Images were acquired one after each other without pause. The focus and exposure was set to visualize post-Golgi carriers. The whole sequence is 20 frames and the movie plays at 0.4 fps. (AVI 2528 kb)

The file contains Supplementary Movie 3.

The file contains Supplementary Movie 3. This movie shows the exit from Golgi of both NCAM-cherry and p75-GFP in a clathrin-depleted MDCK cell starting 10-15 min after release of the 20oC Golgi block. Boxes areas were zoomed below to show a representative post-Golgi carrier containing both NCAM-cherry and p75-GFP. Images were acquired without pause one after another. The focus and exposure was set to visualize post-Golgi carriers. The whole sequence is 20 frames and the movie plays at 0.4 fps. (AVI 2051 kb)

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Deborde, S., Perret, E., Gravotta, D. et al. Clathrin is a key regulator of basolateral polarity. Nature 452, 719–723 (2008). https://doi.org/10.1038/nature06828

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