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Membrane bending by protein–protein crowding

Nature Cell Biology volume 14, pages 944–949 (2012)Cite this article

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Abstract

Curved membranes are an essential feature of dynamic cellular structures, including endocytic pits, filopodia protrusions and most organelles1,2. It has been proposed that specialized proteins induce curvature by binding to membranes through two primary mechanisms: membrane scaffolding by curved proteins or complexes3,4; and insertion of wedge-like amphipathic helices into the membrane5,6. Recent computational studies have raised questions about the efficiency of the helix-insertion mechanism, predicting that proteins must cover nearly 100% of the membrane surface to generate high curvature7,8,9, an improbable physiological situation. Thus, at present, we lack a sufficient physical explanation of how protein attachment bends membranes efficiently. On the basis of studies of epsin1 and AP180, proteins involved in clathrin-mediated endocytosis, we propose a third general mechanism for bending fluid cellular membranes: protein–protein crowding. By correlating membrane tubulation with measurements of protein densities on membrane surfaces, we demonstrate that lateral pressure generated by collisions between bound proteins drives bending. Whether proteins attach by inserting a helix or by binding lipid heads with an engineered tag, protein coverage above ~20% is sufficient to bend membranes. Consistent with this crowding mechanism, we find that even proteins unrelated to membrane curvature, such as green fluorescent protein (GFP), can bend membranes when sufficiently concentrated. These findings demonstrate a highly efficient mechanism by which the crowded protein environment on the surface of cellular membranes can contribute to membrane shape change.

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Figure 1: PtdIns(4,5)P2 concentration in membranes controls the frequency of lipid tubule formation by epsin1.
Figure 2: FLIM–FRET measurements of epsin1 density reveal that PtdIns(4,5)P2 concentration controls protein coverage of membrane surfaces.
Figure 3: Protein coverage controls tubule formation by epsin1 ENTH, regardless of membrane attachment chemistry.
Figure 4: Cryo-electron microscopy reveals that ENTH can drive the formation of highly curved membrane tubules in the absence of its amphipathic helix.
Figure 5: Whenever protein concentration exceeds membrane affinity, high protein coverage drives membrane bending.

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References

  1. Hurley, J. H., Boura, E., Carlson, L. A. & Rozycki, B. Membrane budding. Cell 143, 875–887 (2010).

    Article  CAS  Google Scholar 

  2. Baumgart, T., Capraro, B. R., Zhu, C. & Das, S. L. Thermodynamics and mechanics of membrane curvature generation and sensing by proteins and lipids. Annu. Rev. Phys. Chem. 62, 483–506 (2011).

    Article  CAS  Google Scholar 

  3. Sweitzer, S. M. & Hinshaw, J. E. Dynamin undergoes a GTP-dependent conformational change causing vesiculation. Cell 93, 1021–1029 (1998).

    Article  CAS  Google Scholar 

  4. Peter, B. J. et al. BAR domains as sensors of membrane curvature: the amphiphysin BAR structure. Science 303, 495–499 (2004).

    Article  CAS  Google Scholar 

  5. Ford, M. G. et al. Curvature of clathrin-coated pits driven by epsin. Nature 419, 361–366 (2002).

    Article  CAS  Google Scholar 

  6. Lee, M. C. et al. Sar1p N-terminal helix initiates membrane curvature and completes the fission of a COPII vesicle. Cell 122, 605–617 (2005).

    Article  CAS  Google Scholar 

  7. Campelo, F., McMahon, H. T. & Kozlov, M. M. The hydrophobic insertion mechanism of membrane curvature generation by proteins. Biophys. J. 95, 2325–2339 (2008).

    Article  CAS  Google Scholar 

  8. Blood, P. D., Swenson, R. D. & Voth, G. A. Factors influencing local membrane curvature induction by N-BAR domains as revealed by molecular dynamics simulations. Biophys. J. 95, 1866–1876 (2008).

    Article  CAS  Google Scholar 

  9. Drin, G. & Antonny, B. Amphipathic helices and membrane curvature. FEBS Lett. 584, 1840–1847 (2010).

    Article  CAS  Google Scholar 

  10. Lu, L., Ladinsky, M. S. & Kirchhausen, T. Cisternal organization of the endoplasmic reticulum during mitosis. Mol. Biol. Cell 20, 3471–3480 (2009).

    Article  CAS  Google Scholar 

  11. Wu, M. et al. Coupling between clathrin-dependent endocytic budding and F-BAR-dependent tubulation in a cell-free system. Nat. Cell Biol. 12, 902–908 (2010).

    Article  CAS  Google Scholar 

  12. Schmid, E. M. & McMahon, H. T. Integrating molecular and network biology to decode endocytosis. Nature 448, 883–888 (2007).

    Article  CAS  Google Scholar 

  13. Xie, X., Cho, B. & Fischer, J. A. Drosophila Epsin’s role in Notch ligand cells requires three Epsin protein functions: the lipid binding function of the ENTH domain, a single ubiquitin interaction motif, and a subset of the C-terminal protein binding modules. Dev. Biol. 363, 399–412 (2012).

    Article  CAS  Google Scholar 

  14. Capraro, B. R., Yoon, Y., Cho, W. & Baumgart, T. Curvature sensing by the epsin N-terminal homology domain measured on cylindrical lipid membrane tethers. J. Am. Chem. Soc. 132, 1200–1201 (2010).

    Article  CAS  Google Scholar 

  15. Praefcke, G. J. et al. Evolving nature of the AP2 α-appendage hub during clathrin-coated vesicle endocytosis. EMBO J. 23, 4371–4383 (2004).

    Article  CAS  Google Scholar 

  16. Rossman, J. S., Jing, X., Leser, G. P. & Lamb, R. A. Influenza virus M2 protein mediates ESCRT-independent membrane scission. Cell 142, 902–913 (2010).

    Article  CAS  Google Scholar 

  17. Dibble, C. F. et al. Defining the functional domain of programmed cell death 10 through its interactions with phosphatidylinositol-3,4,5-trisphosphate. PLoS One 5, e11740 (2010).

    Article  Google Scholar 

  18. Fan, W., Nassiri, A. & Zhong, Q. Autophagosome targeting and membrane curvature sensing by Barkor/Atg14(L). Proc. Natl Acad. Sci. USA 108, 7769–7774 (2011).

    Article  CAS  Google Scholar 

  19. Saarikangas, J. et al. Molecular mechanisms of membrane deformation by I-BAR domain proteins. Curr. Biol. 19, 95–107 (2009).

    Article  CAS  Google Scholar 

  20. 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 

  21. Liu, A. P. & Fletcher, D. A. Actin polymerization serves as a membrane domain switch in model lipid bilayers. Biophys. J. 91, 4064–4070 (2006).

    Article  CAS  Google Scholar 

  22. Tsafrir, I. et al. Pearling instabilities of membrane tubes with anchored polymers. Phys. Rev. Lett. 86, 1138–1141 (2001).

    Article  CAS  Google Scholar 

  23. Yoon, Y. et al. Molecular basis of the potent membrane-remodeling activity of the epsin 1 N-terminal homology domain. J. Biol. Chem. 285, 531–540 (2010).

    Article  CAS  Google Scholar 

  24. Feder, J. Random sequential adsorption. J. Theor. Biol. 87, 237–254 (1980).

    Article  CAS  Google Scholar 

  25. Gizeli, E. & Glad, J. Single-step formation of a biorecognition layer for assaying histidine-tagged proteins. Anal. Chem. 76, 3995–4001 (2004).

    Article  CAS  Google Scholar 

  26. Stachowiak, J. C., Hayden, C. C. & Sasaki, D. Y. Steric confinement of proteins on lipid membranes can drive curvature and tubulation. Proc. Natl Acad. Sci. USA 107, 7781–7786 (2010).

    Article  CAS  Google Scholar 

  27. Carnahan, N. F. & Starling, K. E. Equation of state for nonattracting rigid spheres. J. Chem. Phys. 51, 635–636 (1969).

    Article  CAS  Google Scholar 

  28. Kent, M. S. et al. Analysis of myoglobin adsorption to Cu(II)-IDA and Ni(II)-IDA functionalized Langmuir monolayers by grazing incidence neutron and X-ray techniques. Langmuir 20, 2819–2829 (2004).

    Article  CAS  Google Scholar 

  29. Hao, W. et al. Regulation of AP-3 function by inositides. Identification of phosphatidylinositol 3,4,5-trisphosphate as a potent ligand. J. Biol. Chem. 272, 6393–6398 (1997).

    Article  CAS  Google Scholar 

  30. Harrison, S. C. & Kirchhausen, T. Clathrin, cages, and coated vesicles. Cell 33, 650–652 (1983).

    Article  CAS  Google Scholar 

  31. Blondeau, F. et al. Tandem MS analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling. Proc. Natl Acad. Sci. USA 101, 3833–3838 (2004).

    Article  CAS  Google Scholar 

  32. Brady, R. J., Wen, Y. & O’Halloran, T. J. The ENTH and C-terminal domains of Dictyostelium epsin cooperate to regulate the dynamic interaction with clathrin-coated pits. J. Cell Sci. 121, 3433–3444 (2008).

    Article  CAS  Google Scholar 

  33. Dannhauser, P. N. & Ungewickell, E. J. Reconstitution of clathrin-coatedbud and vesicle formation with minimal components. Nat. Cell Biol. 14, 634–639 (2012).

    Article  CAS  Google Scholar 

  34. Engelman, D. M. Membranes are more mosaic than fluid. Nature 438, 578–580 (2005).

    Article  CAS  Google Scholar 

  35. Yuste, S. B. & A, S. A heuristic radial distribution function for hard disks. J. Chem. Phys. 99, 2020–2023 (1993).

    Article  CAS  Google Scholar 

  36. Pack, D. W. & Arnold, F. H. Langmuir monolayer characterization of metal chelating lipids for protein targeting to membranes. Chem. Phys. Lipids 86, 135–152 (1997).

    Article  CAS  Google Scholar 

  37. Angelova, M. & Dimitrov, D. Liposome electroformation. Faraday Dis. 81, 303–311 (1986).

    Article  CAS  Google Scholar 

  38. Carvalho, K., Ramos, L., Roy, C. & Picart, C. Giant unilamellar vesicles containing phosphatidylinositol(4,5)bisphosphate: characterization and functionality. Biophys. J. 95, 4348–4360 (2008).

    Article  CAS  Google Scholar 

  39. Sherman, M. B. et al. Removal of divalent cations induces structural transitions in red clover necrotic mosaic virus, revealing a potential mechanism for RNA release. J. Virol. 80, 10395–10406 (2006).

    Article  CAS  Google Scholar 

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Acknowledgements

We acknowledge H. McMahon (LMB, Cambridge, UK) and M. Ford (UC Davis, USA) for discussions on this work and contribution of reagents. This work was supported by the US Department of Energy, Office of Basic Energy Sciences, Division of Materials Science and Engineering (membrane studies and development, J.C.S., D.Y.S., Sandia), DEAC02-05CH11231 (membrane modelling, P.L.G., C.J.R.) and Division of Chemical Sciences, Geosciences, and Biosciences (fluorescence imaging and analysis, C.C.H., Sandia); as well as the Laboratory Directed Research and Development program at Sandia National Laboratories (engineered vesicle design, J.C.S., D.Y.S., C.C.H., D.A.F.); the NIH NIGMS and Nanomedicine Development Centers (protein membrane interactions, D.A.F., E.M.S., H.S.A.); a Sealy and Smith Foundation grant to the Sealy Center for Structural Biology and Molecular Biophysics (cryo-electron microscopy facility, M.B.S.); and the Miller Institute for Basic Research in Science (E.M.S.). Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy’s National Nuclear Security Administration under contract DE-AC04-94AL85000.

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Author notes

  1. Jeanne C. Stachowiak and Eva M. Schmid: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Biomedical Engineering, The University of Texas at Austin, Austin, Texas 78712, USA

    Jeanne C. Stachowiak

  2. Sandia National Laboratories, Livermore, California 94551, USA

    Jeanne C. Stachowiak, Darryl Y. Sasaki & Carl C. Hayden

  3. Department of Bioengineering, University of California, Berkeley, California 94720, USA

    Eva M. Schmid, Hyoung Sook Ann & Daniel A. Fletcher

  4. Biophysics Gradate Group, University of California, Berkeley, California 94720, USA

    Christopher J. Ryan, Phillip L. Geissler & Daniel A. Fletcher

  5. Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555, USA

    Michael B. Sherman

  6. Department of Chemistry, University of California, Berkeley, California 94720, USA

    Phillip L. Geissler

  7. Chemical Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA

    Phillip L. Geissler

  8. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA

    Daniel A. Fletcher

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Contributions

J.C.S., E.M.S., M.B.S. and C.C.H. performed experiments. E.M.S., H.S.A. and D.Y.S. created unique materials. C.J.R., P.L.G. and C.C.H. performed simulations and modelling. All authors designed experiments. J.C.S., E.M.S., C.J.R., D.A.F. and C.C.H. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Jeanne C. Stachowiak, Eva M. Schmid, Daniel A. Fletcher or Carl C. Hayden.

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The authors declare no competing financial interests.

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Stachowiak, J., Schmid, E., Ryan, C. et al. Membrane bending by protein–protein crowding. Nat Cell Biol 14, 944–949 (2012). https://doi.org/10.1038/ncb2561

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