Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells

Nature Cell Biology volume 8, pages 46–54 (2006)Cite this article

Abstract

Previous studies provide evidence for an endocytic mechanism in mammalian cells that is distinct from both clathrin-coated pits and caveolae1,2,3,4,5, and is not inhibited by overexpression of GTPase-null dynamin mutants1,2,3,4,6. This mechanism, however, has been defined largely in these negative terms. We applied a ferro-fluid-based purification of endosomes to identify endosomal proteins. One of the proteins identified in this way was flotillin-1 (also called reggie-2)7,8. Here, we show that flotillin-1 resides in punctate structures within the plasma membrane and in a specific population of endocytic intermediates. These intermediates accumulate both glycosylphosphatidylinositol (GPI)-linked proteins and cholera toxin B subunit4,9. Endocytosis in flotillin-1-containing intermediates is clathrin-independent. Total internal reflection microscopy and immuno-electron microscopy revealed that flotillin-1-containing regions of the plasma membrane seem to bud into the cell, and are distinct from clathrin-coated pits and caveolin-1-positive caveolae10. Flotillin-1 small interfering RNA (siRNA) inhibited both clathrin-independent uptake of cholera toxin and endocytosis of a GPI-linked protein. We propose that flotillin-1 is one determinant of a clathrin-independent endocytic pathway in mammalian cells.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Flotillin-1 is found in endosomes and does not colocalize with transferrin or caveolin-1.
Figure 2: Flotillin-1 defines a class of early endosomal intermediates that do not contain caveolin-1 or transferrin.
Figure 3: Flotillin-1 defines dynamic plasma-membrane microdomains that appear to bud into the cell.
Figure 4: Flotillin-1 siRNAs inhibit clathrin-independent endocytosis.
Figure 5: Combinatorial effects of flotillin-1 siRNA and dynamin-2–K44A.

Similar content being viewed by others

References

  1. Kirkham, M. et al. Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles. J. Cell Biol. 168, 465–476 (2005).

    Article  CAS  Google Scholar 

  2. Lamaze, C. et al. Interleukin 2 receptors and detergent-resistant membrane domains define a clathrin-independent endocytic pathway. Mol. Cell 7, 661–671 (2001).

    Article  CAS  Google Scholar 

  3. Naslavsky, N., Weigert, R. & Donaldson, J. G. Characterization of a nonclathrin endocytic pathway: membrane cargo and lipid requirements. Mol. Biol. Cell 15, 3542–3552 (2004).

    Article  CAS  Google Scholar 

  4. Sabharanjak, S., Sharma, P., Parton, R. G. & Mayor, S. GPI-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway. Dev. Cell 2, 411–423 (2002).

    Article  CAS  Google Scholar 

  5. Nichols, B. J. et al. Rapid cycling of lipid raft markers between the cell surface and Golgi complex. J. Cell Biol. 153, 529–541 (2001).

    Article  CAS  Google Scholar 

  6. Puri, V. et al. Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways. J. Cell Biol. 154, 535–547 (2001).

    Article  CAS  Google Scholar 

  7. Lang, D. M. et al. Identification of reggie-1 and reggie-2 as plasma membrane-associated proteins which cocluster with activated GPI-anchored cell adhesion molecules in non-caveolar micropatches in neurons. J. Neurobiol. 37, 502–523 (1998).

    Article  CAS  Google Scholar 

  8. Bickel, P. E. et al. Flotillin and epidermal surface antigen define a new family of caveolae-associated integral membrane proteins. J. Biol. Chem. 272, 13793–13802 (1997).

    Article  CAS  Google Scholar 

  9. Nichols, B. J. GM1-containing lipid rafts are depleted within clathrin-coated pits. Curr. Biol. 13, 686–690 (2003).

    Article  CAS  Google Scholar 

  10. Conner, S. D. & Schmid, S. L. Regulated portals of entry into the cell. Nature 422, 37–44 (2003).

    Article  CAS  Google Scholar 

  11. Nolta, K. V., Rodriguez-Paris, J. M. & Steck, T. L. Analysis of successive endocytic compartments isolated from Dictyostelium discoideum by magnetic fractionation. Biochim. Biophys. Acta 1224, 237–246 (1994).

    Article  CAS  Google Scholar 

  12. Rajendran, L. et al. Asymmetric localization of flotillins/reggies in preassembled platforms confers inherent polarity to hematopoietic cells. Proc. Natl Acad. Sci. USA 100, 8241–8246 (2003).

    Article  CAS  Google Scholar 

  13. Morrow, I. C. et al. Flotillin-1/reggie-2 traffics to surface raft domains via a novel golgi-independent pathway. Identification of a novel membrane targeting domain and a role for palmitoylation. J. Biol. Chem. 277, 48834–48841 (2002).

    Article  CAS  Google Scholar 

  14. Liu, J., Deyoung, S. M., Zhang, M., Dold, L. H. & Saltiel, A. R. The SPFH domain of flotillin-1 contains distinct sequences that direct plasma membrane localization and protein interactions in 3T3-L1 adipocytes. J. Biol. Chem. 280, 16125–16134 (2005).

    Article  CAS  Google Scholar 

  15. Kokubo, H. et al. Ultrastructural localization of flotillin-1 to cholesterol-rich membrane microdomains, rafts, in rat brain tissue. Brain Res. 965, 83–90 (2003).

    Article  CAS  Google Scholar 

  16. Volonte, D. et al. Flotillins/cavatellins are differentially expressed in cells and tissues and form a hetero-oligomeric complex with caveolins in vivo. Characterization and epitope-mapping of a novel flotillin-1 monoclonal antibody probe. J. Biol. Chem. 274, 12702–12709 (1999).

    Article  CAS  Google Scholar 

  17. Santamaria, A. et al. PTOV1 enables the nuclear translocation and mitogenic activity of flotillin-1, a major protein of lipid rafts. Mol. Cell. Biol. 25, 1900–1911 (2005).

    Article  CAS  Google Scholar 

  18. Pelkmans, L., Burli, T., Zerial, M. & Helenius, A. Caveolin-stabilized membrane domains as multifunctional transport and sorting devices in endocytic membrane traffic. Cell 118, 767–780 (2004).

    Article  CAS  Google Scholar 

  19. Parton, R. G. & Richards, A. A. Lipid rafts and caveolae as portals for endocytosis: new insights and common mechanisms. Traffic 4, 724–738 (2003).

    Article  CAS  Google Scholar 

  20. Nichols, B. J. A distinct class of endosome mediates clathrin-independent endocytosis to the Golgi complex. Nature Cell Biol. 4, 374–378 (2002).

    Article  CAS  Google Scholar 

  21. Sandvig, K. et al. Pathways followed by protein toxins into cells. Int. J. Med. Microbiol. 293, 483–490 (2004).

    Article  CAS  Google Scholar 

  22. Orlandi, P. A. & Fishman, P. H. Filipin-dependent inhibition of cholera toxin: evidence for toxin internalization and activation through caveolae-like domains. J. Cell. Biol. 141, 905–915 (1998).

    Article  CAS  Google Scholar 

  23. Ford, M. G. et al. Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes. Science 291, 1051–1055 (2001).

    Article  CAS  Google Scholar 

  24. Merrifield, C. J., Perrais, D. & Zenisek, D. Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells. Cell 121, 593–606 (2005).

    Article  CAS  Google Scholar 

  25. Pelkmans, L., Kartenbeck, J. & Helenius, A. Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER. Nature Cell Biol. 3, 473–483 (2001).

    Article  CAS  Google Scholar 

  26. Henley, J. R., Krueger, E. W., Oswald, B. J. & McNiven, M. A. Dynamin-mediated internalization of caveolae. J. Cell Biol. 141, 85–99 (1998).

    Article  CAS  Google Scholar 

  27. Oh, P., McIntosh, D. P. & Schnitzer, J. E. Dynamin at the neck of caveolae mediates their budding to form transport vesicles by GTP-driven fission from the plasma membrane of endothelium. J. Cell Biol. 141, 101–114 (1998).

    Article  CAS  Google Scholar 

  28. Damke, H., Baba, T., van der Bliek, A. M. & Schmid, S. L. Clathrin-independent pinocytosis is induced in cells overexpressing a temperature-sensitive mutant of dynamin. J. Cell Biol. 131, 69–80 (1995).

    Article  CAS  Google Scholar 

  29. Damm, E. M. et al. Clathrin- and caveolin-1-independent endocytosis: entry of simian virus 40 into cells devoid of caveolae. J. Cell Biol. 168, 477–488 (2005).

    Article  CAS  Google Scholar 

  30. Mayor, S. & Riezman, H. Sorting GPI-anchored proteins. Nature Rev. Mol. Cell Biol. 5, 110–120 (2004).

    Article  CAS  Google Scholar 

  31. Nichols, B. Caveosomes and endocytosis of lipid rafts. J. Cell Sci. 116, 4707–4714 (2003).

    Article  CAS  Google Scholar 

  32. Brown, F. D., Rozelle, A. L., Yin, H. L., Balla, T. & Donaldson, J. G. Phosphatidylinositol 4,5-bisphosphate and Arf6-regulated membrane traffic. J. Cell Biol. 154, 1007–1017 (2001).

    Article  CAS  Google Scholar 

  33. Fisher, J. A., Girdler, G. & Khakh, B. S. Time-resolved measurement of state-specific P2X2 ion channel cytosolic gating motions. J. Neurosci. 24, 10475–10487 (2004).

    Article  CAS  Google Scholar 

  34. Bright, N. A., Reaves, B. J., Mullock, B. M. & Luzio, J. P. Dense core lysosomes can fuse with late endosomes and are re-formed from the resultant hybrid organelles. J. Cell Sci. 110, 2027–2040 (1997).

    CAS  PubMed  Google Scholar 

  35. Slot, J. W., Geuze, H. J., Gigengack, S., Lienhard, G. E. & James, D. E. Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat. J. Cell Biol. 113, 123–135 (1991).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

H. Pelham and S. Munro provided criticism of the manuscript. S.-Y. Peak-Chew and F. Begum carried out protein identification by mass spectrometry. D. Bowser and B. Khakh set up the TIR microscope and assisted with its use. J. Skepper carried out preparation of samples for electron microscopy and assisted in their analysis.

Author information

Authors and Affiliations

  1. MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK

    Oleg O. Glebov & Benjamin J. Nichols

  2. Cambridge Institute of Medical Research, Hills Road, Cambridge, CB2 2XY, UK

    Nicholas A. Bright

Authors
  1. Oleg O. Glebov
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nicholas A. Bright
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Benjamin J. Nichols
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Benjamin J. Nichols.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary figures S1, S2, S3 and S4 (PDF 384 kb)

Supplementary Information

Supplementary Movie s1 (MOV 3484 kb)

Supplementary Information

Supplementary Movie s2 (MOV 271 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Glebov, O., Bright, N. & Nichols, B. Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells. Nat Cell Biol 8, 46–54 (2006). https://doi.org/10.1038/ncb1342

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb1342

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing