Abstract

Membrane interfaces formed at cell–cell junctions are associated with characteristic patterns of membrane proteins whose organization is critical for intracellular signalling. To isolate the role of membrane protein size in pattern formation, we reconstituted model membrane interfaces in vitro using giant unilamellar vesicles decorated with synthetic binding and non-binding proteins. We show that size differences between membrane proteins can drastically alter their organization at membrane interfaces, with as little as a 5 nm increase in non-binding protein size driving its exclusion from the interface. Combining in vitro measurements with Monte Carlo simulations, we find that non-binding protein exclusion is also influenced by lateral crowding, binding protein affinity, and thermally driven membrane height fluctuations that transiently limit access to the interface. This sensitive and highly effective means of physically segregating proteins has implications for cell–cell contacts such as T-cell immunological synapses (for example, CD45 exclusion) and epithelial cell junctions (for example, E-cadherin enrichment), as well as for protein sorting at intracellular contact points between membrane-bound organelles.

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References

  1. 1.

    Models for the specific adhesion of cells to cells. Science 200, 618–627 (1978).

  2. 2.

    The immunological synapse. Arthritis Res. Ther. 4, S119–S125 (2002).

  3. 3.

    , , & Myoblast fusion: when it takes more to make one. Dev. Biol. 341, 66–83 (2010).

  4. 4.

    , , & Mechanisms of epithelial cell–cell adhesion and cell compaction revealed by high-resolution tracking of E-cadherin-green fluorescent protein. J. Cell Biol. 142, 1105–1119 (1998).

  5. 5.

    et al. Activation of the innate immune receptor Dectin-1 upon formation of a ‘phagocytic synapse’. Nature 472, 471–475 (2011).

  6. 6.

    et al. Structure of a tyrosine phosphatase adhesive interaction reveals a spacer-clamp mechanism. Science 317, 1217–1220 (2007).

  7. 7.

    , , , & Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395, 82–86 (1998).

  8. 8.

    Making a little affinity go a long way: a topological view of LFA-1 regulation. Cell Adhes. Commun. 6, 255–262 (1998).

  9. 9.

    et al. The immunological synapse: a molecular machine controlling T cell activation. Science 285, 221–227 (1999).

  10. 10.

    et al. T cell receptor ligation induces the formation of dynamically regulated signaling assemblies. J. Cell Biol. 158, 1263–1275 (2002).

  11. 11.

    , , , & T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster. Immunity 25, 117–127 (2006).

  12. 12.

    & Biophysical mechanism of T-cell receptor triggering in a reconstituted system. Nature 487, 64–69 (2012).

  13. 13.

    et al. The large ectodomains of CD45 and CD148 regulate their segregation from and inhibition of ligated T-cell receptor. Blood 121, 4295–4302 (2013).

  14. 14.

    , & The biology of the T-cell antigen receptor and its role in the skin immune system. J. Invest. Dermatol. 94, 91S–100S (1990).

  15. 15.

    & CD2: an exception to the immunoglobulin superfamily concept? Science 273, 1241–1242 (1996).

  16. 16.

    , , , & T-cell receptor triggering is critically dependent on the dimensions of its peptide-MHC ligand. Nature 436, 578–582 (2005).

  17. 17.

    , , & Spatial organization of transmembrane receptor signalling. EMBO J. 29, 2677–2688 (2010).

  18. 18.

    et al. Phase transitions in the assembly of multivalent signalling proteins. Nature 483, 336–340 (2012).

  19. 19.

    , & Continuous membrane-cytoskeleton adhesion requires continuous accommodation to lipid and cytoskeleton dynamics. Annu. Rev. Biophys. Biomol. Struct. 35, 417–434 (2006).

  20. 20.

    , , , & Adhesion of membranes via receptor–ligand complexes: domain formation, binding cooperativity, and active processes. Soft Matter 5, 3213–3224 (2009).

  21. 21.

    et al. Nanoscale increases in CD2-CD48-mediated intermembrane spacing decrease adhesion and reorganize the immunological synapse. J. Biol. Chem. 283, 34414–34422 (2008).

  22. 22.

    et al. Mechanisms for size-dependent protein segregation at immune synapses assessed with molecular rulers. Biophys. J. 100, 2865–2874 (2011).

  23. 23.

    , & Expression of soluble isoforms of rat CD45. Analysis by electron microscopy and use in epitope mapping of anti-CD45R monoclonal antibodies. Immunology 76, 310–317 (1992).

  24. 24.

    , & Segregation of receptor–ligand complexes in cell adhesion zones: phase diagrams and the role of thermal membrane roughness. New J. Phys. 12, 095003 (2010).

  25. 25.

    et al. Boltzmann energy-based image analysis demonstrates that extracellular domain size differences explain protein segregation at immune synapses. PLoS Comput. Biol. 7, e1002076–11 (2011).

  26. 26.

    , & Binding constants of membrane-anchored receptors and ligands depend strongly on the nanoscale roughness of membranes. Proc. Natl Acad. Sci. USA 110, 15283–15288 (2013).

  27. 27.

    Structure and dynamics of green fluorescent protein. Curr. Opin. Struct. Biol. 7, 821–827 (1997).

  28. 28.

    CLONTECH Laboratories, Inc. Living Colors, Clontech User Manual 1–51 (1998).

  29. 29.

    , & Structural and thermodynamic correlates of T cell signaling. Annu. Rev. Biophys. Biomol. Struct. 31, 121–149 (2002).

  30. 30.

    , & Density of newly synthesized plasma membrane proteins in intracellular membranes II. Biochemical studies. J. Cell Biol. 98, 2142–2147 (1984).

  31. 31.

    et al. Molecular anatomy of a trafficking organelle. Cell 127, 831–846 (2006).

  32. 32.

    , & New modeling of reflection interference contrast microscopy including polarization and numerical aperture effects: application to nanometric distance measurements and object profile reconstruction. Langmuir 26, 1940–1948 (2010).

  33. 33.

    , & The molecular structure of green fluorescent protein. Nature Biotechnol. 14, 1246–1251 (1996).

  34. 34.

    , , & Line tension and stability of domains in cell-adhesion zones mediated by long and short receptor-ligand complexes. PLoS ONE 6, e23284 (2011).

  35. 35.

    , , , & Transforming binding affinities from three dimensions to two with application to cadherin clustering. Nature 475, 510–513 (2011).

  36. 36.

    , & A new mechanism of model membrane fusion determined from Monte Carlo simulation. Biophys. J. 85, 1611–1623 (2003).

  37. 37.

    & Simulations of fluid self-avoiding membranes. Europhys. Lett. 12, 295–300 (1990).

  38. 38.

    & Network models of fluid, hexatic and polymerized membranes. J. Phys. Condens. Matter 9, 8795–8834 (1997).

  39. 39.

    & Immunoglobulin superfamily proteins in Caenorhabditis elegans. J. Mol. Biol. 296, 1367–1383 (2000).

  40. 40.

    The immunoglobulin superfamily in Drosophila melanogaster and Caenorhabditis elegans and the evolution of complexity. Dev. Camb. Engl. 130, 6317–6328 (2003).

  41. 41.

    et al. Organization and function of membrane contact sites. Biochim. Biophys. Acta 1833, 2526–2541 (2013).

  42. 42.

    The molecular hug between the ER and the mitochondria. Curr. Opin. Cell Biol. 25, 443–448 (2013).

  43. 43.

    & Mechanisms of membrane fusion: disparate players and common principles. Nature Rev. Mol. Cell Biol. 9, 543–556 (2008).

  44. 44.

    & Liposome electroformation. Faraday Discuss. Chem. Soc. 81, 303–311 (1986).

  45. 45.

    & Kinetic control of histidine-tagged protein surface density on supported lipid bilayers. Langmuir 24, 4145–4149 (2008).

  46. 46.

    , , & Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002).

  47. 47.

    & Fluorescence correlation spectroscopy: the technique and its applications. Rep. Prog. Phys. 65, 251–297 (2002).

  48. 48.

    Random surface discretizations and the renormalization of the bending rigidity. J. Phys. Fr. 6, 1305–1320 (1996).

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Acknowledgements

We acknowledge R. Vale and C. Peel for helpful discussions. This work was supported by a Graduate Fellows Research Program grant from the National Science Foundation (NSF) for M.H.B.; a Cancer Research Institute Post-Doctoral Fellowship and a K99 grant from the National Institute of Health (NIH, K99AI093884 and R00AI093884) for K.C.; a Forschungsstipendium of the Deutsche Forschungsgemeinschaft (DFG grant no. We 5004/2) for J.W.; a NIH grant (R37AI043542), a NIGMS Nanomedicine Development Center grant (PN2EY016586) and a Wellcome Trust Principal Research Fellowship to M.L.D.; and a NIH Nanomedicine Development Center grant (PN2EY016546) and an NIH R01 grant (GM114344) to D.A.F. This research was also supported by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, US Department of Energy, FWP number SISGRKN.

Author information

Author notes

    • Eva M. Schmid
    •  & Matthew H. Bakalar

    These authors contributed equally to this work.

    • Kaushik Choudhuri
    •  & Hyoung Sook Ann

    Present addresses: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA (K.C.); Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Champaign, Illinois 61820, USA (H.S.A.).

Affiliations

  1. Department of Bioengineering & Biophysics Program, University of California, Berkeley, California 94720, USA

    • Eva M. Schmid
    • , Matthew H. Bakalar
    • , Hyoung Sook Ann
    •  & Daniel A. Fletcher
  2. UC Berkeley/UC San Francisco Graduate Group in Bioengineering, Berkeley, California 94720, USA

    • Matthew H. Bakalar
    •  & Daniel A. Fletcher
  3. Skirball Institute, New York University School of Medicine, New York, New York 10016, USA

    • Kaushik Choudhuri
    •  & Michael L. Dustin
  4. Department of Chemistry, University of California, Berkeley, California 94720, USA

    • Julian Weichsel
    •  & Phillip L. Geissler
  5. Chemical Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA

    • Phillip L. Geissler
  6. Kennedy Institute, NDORMS, University of Oxford, Oxford OX3 7DL, UK

    • Michael L. Dustin
  7. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA

    • Daniel A. Fletcher

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Contributions

All authors contributed to design of the experiments. E.M.S. and M.H.B. performed experiments. E.M.S., H.S.A. and M.H.B. created unique materials. J.W., M.H.B. and P.L.G. performed simulations and modelling. E.M.S., M.H.B. and D.A.F. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Eva M. Schmid or Matthew H. Bakalar or Daniel A. Fletcher.

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DOI

https://doi.org/10.1038/nphys3678