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Arf6 and microtubules in adhesion-dependent trafficking of lipid rafts

Nature Cell Biology volume 9, pages 1381–1391 (2007)Cite this article

Abstract

Integrin-mediated adhesion regulates membrane binding sites for Rac1 within lipid rafts. Detachment of cells from the substratum triggers the clearance of rafts from the plasma membrane through caveolin-dependent internalization. The small GTPase Arf6 and microtubules also regulate Rac-dependent cell spreading and migration, but the mechanisms are poorly understood. Here we show that endocytosis of rafts after detachment requires F-actin, followed by microtubule-dependent trafficking to recycling endosomes. When cells are replated on fibronectin, rafts exit from recycling endosomes in an Arf6-dependent manner and return to the plasma membrane along microtubules. Both of these steps are required for the plasma membrane targeting of Rac1 and for its activation. These data therefore define a new membrane raft trafficking pathway that is crucial for anchorage-dependent signalling.

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Figure 1: The cytoskeleton in raft endocytosis.
Figure 2: Endocytosed rafts do not localize to the Golgi or the SER.
Figure 3: Endocytosed rafts target to recycling endosomes.
Figure 4: Inhibition of Arf6 function.
Figure 5: Adhesion-dependent activation of Arf6 promotes raft exocytosis.
Figure 6: Microtubules in raft exocytosis.
Figure 7: Cell detachment triggers actin-dependent endocytosis of rafts through caveolae.

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Acknowledgements

We thank Robert Nabi (University of British Colombia, Vancouver, Canada) for generously providing anti-AMF antibody; Richard Kahn (Emory University School of Medicine, Atlanta GA) for antibodies to Arfs 1, 3, 4 and 5; Jennifer Lippincott-Schwartz (NICHHD, NIH, Bethesda, MD) for VSVG-GEP; and Vivek Malhotra (University of California, San Diego, CA) for PKD constructs. Anti-tubulin antibody was obtained from the Developmental Studies Hybridoma Bank. This work was supported by a US Public Health Service grant RO1 GM47214 to M.A.S.

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Authors and Affiliations

  1. Robert M. Beirne Cardiovascular Research Center, Mellon Prostate Cancer Research Institute, University of Virginia, Charlottesville, 22908, Virginia, USA

    Nagaraj Balasubramanian, David W. Scott & Martin Alexander Schwartz

  2. Department of Cell Biology, Mellon Prostate Cancer Research Institute, University of Virginia, Charlottesville, 22908, Virginia, USA

    J. David Castle & James E. Casanova

  3. Departments of Microbiology and Biomedical Engineering, Mellon Prostate Cancer Research Institute, University of Virginia, Charlottesville, 22908, Virginia, USA

    Martin Alexander Schwartz

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Contributions

N.B. designed and carried out experiments, analysed data and wrote the manuscript. D.W.S. carried out experiments. J.D.C. and J.E.C. provided reagents and advice. M.A.S. designed experiments, analysed data and wrote the manuscript.

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Correspondence to Martin Alexander Schwartz.

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Balasubramanian, N., Scott, D., Castle, J. et al. Arf6 and microtubules in adhesion-dependent trafficking of lipid rafts. Nat Cell Biol 9, 1381–1391 (2007). https://doi.org/10.1038/ncb1657

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