Advertisement

Metabolically Biotinylated Reporters for Electron Microscopic Imaging of Cytoplasmic Membrane Microdomains

  • Kimberly J. Krager
  • John G. KolandEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1376)

Abstract

The protein and lipid substituents of cytoplasmic membranes are not in general homogeneously distributed across the membrane surface. Many membrane proteins, including ion channels, receptors, and other signaling molecules, exhibit a profound submicroscopic spatial organization, in some cases clustering in submicron membrane subdomains having a protein and lipid composition distinct from that of the bulk membrane. In the case of membrane-associated signaling molecules, mounting evidence indicates that their nanoscale organization, for example the colocalization of differing signaling molecules in the same membrane microdomains versus their segregation into distinct microdomain species, can significantly impact signal transduction. Biochemical membrane fractionation approaches have been used to characterize membrane subdomains of unique protein and lipid composition, including cholesterol-rich lipid raft structures. However, the intrinsically perturbing nature of fractionation methods makes the interpretation of such characterization subject to question, and indeed the existence and significance of lipid rafts remain controversial. Electron microscopic (EM) imaging of immunogold-labeled proteins in plasma membrane sheets has emerged as a powerful method for visualizing the nanoscale organization and colocalization of membrane proteins, which is not as perturbing of membrane structure as are biochemical approaches. For the purpose of imaging putative lipid raft structures, we recently developed a streamlined EM membrane sheet imaging procedure that employs a unique genetically encoded and metabolically biotinylated reporter that is targeted to membrane inner leaflet lipid rafts. We describe here the principles of this procedure and its application in the imaging of plasma membrane inner leaflet lipid rafts.

Key words

Membrane protein Membrane microdomain Nanodomain Lipid raft Electron microscopy EM imaging Avidin-biotin detection Gold labeling 

Notes

Acknowledgement

The authors wish to acknowledge the University of Iowa Central Microscopy Research Facility for their expert technical support and guidance, and for their provision of instrumentation used in TEM imaging.

References

  1. 1.
    Grakoui A, Bromley SK, Sumen C, Davis MM, Shaw AS, Allen PM, Dustin ML (1999) The immunological synapse: a molecular machine controlling T cell activation. Science 285:221–227PubMedCrossRefGoogle Scholar
  2. 2.
    Chichili GR, Rodgers W (2007) Clustering of membrane raft proteins by the actin cytoskeleton. J Biol Chem 282:36682–36691PubMedCrossRefGoogle Scholar
  3. 3.
    Lajoie P, Partridge EA, Guay G, Goetz JG, Pawling J, Lagana A, Joshi B, Dennis JW, Nabi IR (2007) Plasma membrane domain organization regulates EGFR signaling in tumor cells. J Cell Biol 179:341–356PubMedPubMedCentralCrossRefGoogle Scholar
  4. 4.
    Nicolau DV Jr, Burrage K, Parton RG, Hancock JF (2006) Identifying optimal lipid raft characteristics required to promote nanoscale protein-protein interactions on the plasma membrane. Mol Cell Biol 26:313–323PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Lagerholm BC, Weinreb GE, Jacobson K, Thompson NL (2005) Detecting microdomains in intact cell membranes. Annu Rev Phys Chem 56:309–336PubMedCrossRefGoogle Scholar
  6. 6.
    Pike LJ, Han X, Gross RW (2005) Epidermal growth factor receptors are localized to lipid rafts that contain a balance of inner and outer leaflet lipids: a shotgun lipidomics study. J Biol Chem 280:26796–26804PubMedPubMedCentralCrossRefGoogle Scholar
  7. 7.
    Pyenta PS, Holowka D, Baird B (2001) Cross-correlation analysis of inner-leaflet-anchored green fluorescent protein co-redistributed with IgE receptors and outer leaflet lipid raft components. Biophys J 80:2120–2132PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Keating E, Nohe A, Petersen NO (2008) Studies of distribution, location and dynamic properties of EGFR on the cell surface measured by image correlation spectroscopy. Eur Biophys J 37:469–481PubMedCrossRefGoogle Scholar
  9. 9.
    Orr G, Hu D, Ozcelik S, Opresko LK, Wiley HS, Colson SD (2005) Cholesterol dictates the freedom of EGF receptors and HER2 in the plane of the membrane. Biophys J 89:1362–1373PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Xiao Z, Zhang W, Yang Y, Xu L, Fang X (2008) Single-molecule diffusion study of activated EGFR implicates its endocytic pathway. Biochem Biophys Res Commun 369:730–734PubMedCrossRefGoogle Scholar
  11. 11.
    Kawashima N, Nakayama K, Itoh K, Itoh T, Ishikawa M, Biju V (2010) Reversible dimerization of EGFR revealed by single-molecule fluorescence imaging using quantum dots. Chemistry 16:1186–1192PubMedCrossRefGoogle Scholar
  12. 12.
    Waugh MG, Lawson D, Hsuan JJ (1999) Epidermal growth factor receptor activation is localized within low-buoyant density, non-caveolar membrane domains. Biochem J 337:591–597PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Ringerike T, Blystad FD, Levy FO, Madshus IH, Stang E (2002) Cholesterol is important in control of EGF receptor kinase activity but EGF receptors are not concentrated in caveolae. J Cell Sci 115:1331–1340PubMedGoogle Scholar
  14. 14.
    Roepstorff K, Thomsen P, Sandvig K, van Deurs B (2002) Sequestration of epidermal growth factor receptors in non-caveolar lipid rafts inhibits ligand binding. J Biol Chem 277:18954–18960PubMedCrossRefGoogle Scholar
  15. 15.
    Prior IA, Parton RG, Hancock JF (2003) Observing cell surface signaling domains using electron microscopy. Sci STKE 2003:PL9PubMedGoogle Scholar
  16. 16.
    Sanan DA, Anderson RG (1991) Simultaneous visualization of LDL receptor distribution and clathrin lattices on membranes torn from the upper surface of cultured cells. J Histochem Cytochem 39:1017–1024PubMedCrossRefGoogle Scholar
  17. 17.
    Prior IA, Muncke C, Parton RG, Hancock JF (2003) Direct visualization of Ras proteins in spatially distinct cell surface microdomains. J Cell Biol 160:165–170PubMedPubMedCentralCrossRefGoogle Scholar
  18. 18.
    Wilson BS, Pfeiffer JR, Oliver JM (2002) FcepsilonRI signaling observed from the inside of the mast cell membrane. Mol Immunol 38:1259–1268PubMedCrossRefGoogle Scholar
  19. 19.
    Yang S, Raymond-Stintz MA, Ying W, Zhang J, Lidke DS, Steinberg SL, Williams L, Oliver JM, Wilson BS (2007) Mapping ErbB receptors on breast cancer cell membranes during signal transduction. J Cell Sci 120:2763–2773PubMedCrossRefGoogle Scholar
  20. 20.
    Wilson BS, Steinberg SL, Liederman K, Pfeiffer JR, Surviladze Z, Zhang J, Samelson LE, Yang LH, Kotula PG, Oliver JM (2004) Markers for detergent-resistant lipid rafts occupy distinct and dynamic domains in native membranes. Mol Biol Cell 15:2580–2592PubMedPubMedCentralCrossRefGoogle Scholar
  21. 21.
    Krager KJ, Sarkar M, Twait EC, Lill NL, Koland JG (2012) A novel biotinylated lipid raft reporter for electron microscopic imaging of plasma membrane microdomains. J Lipid Res 53:2214–2225PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    Zlatkine P, Mehul B, Magee AI (1997) Retargeting of cytosolic proteins to the plasma membrane by the Lck protein tyrosine kinase dual acylation motif. J Cell Sci 110(Pt 5):673–679PubMedGoogle Scholar
  23. 23.
    Johnson SA, Stinson BM, Go MS, Carmona LM, Reminick JI, Fang X, Baumgart T (2010) Temperature-dependent phase behavior and protein partitioning in giant plasma membrane vesicles. Biochim Biophys Acta 1798:1427–1435PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    Parrott MB, Barry MA (2000) Metabolic biotinylation of recombinant proteins in mammalian cells and in mice. Mol Ther 1:96–104PubMedPubMedCentralCrossRefGoogle Scholar
  25. 25.
    Tannous BA, Grimm J, Perry KF, Chen JW, Weissleder R, Breakefield XO (2006) Metabolic biotinylation of cell surface receptors for in vivo imaging. Nat Methods 3:391–396PubMedCrossRefGoogle Scholar
  26. 26.
    Haase P (2001) Can isotropy vs. anisotropy in the spatial association of plant species reveal physical vs. biotic facilitation? J Veg Sci 12:127–136CrossRefGoogle Scholar
  27. 27.
    Kiskowski MA, Hancock JF, Kenworthy AK (2009) On the use of Ripley’s K-function and its derivatives to analyze domain size. Biophys J 97:1095–1103PubMedPubMedCentralCrossRefGoogle Scholar
  28. 28.
    Haase P (1995) Spatial pattern analysis in ecology based on Ripley’s K-function: introduction and methods of edge correction. J Veg Sci 6:575–582CrossRefGoogle Scholar
  29. 29.
    Wilson BS, Pfeiffer JR, Oliver JM (2000) Observing FcepsilonRI signaling from the inside of the mast cell membrane. J Cell Biol 149:1131–1142PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of PharmacologyThe University of Iowa, Carver College of MedicineIowa CityUSA
  2. 2.Division of Radiation HealthUniversity of Arkansas for Medical Sciences, College of PharmacyLittle RockUSA

Personalised recommendations