Caveolae pp 11-25 | Cite as

Freeze-Fracture Replica Immunolabeling of Cryopreserved Membrane Compartments, Cultured Cells and Tissues

  • Eric Seemann
  • Michael M. KesselsEmail author
  • Britta QualmannEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2169)


Membrane topology information and views of membrane-embedded protein complexes promote our understanding of membrane organization and cell biological function involving membrane compartments. Freeze-fracturing of biological membranes offers both stunning views onto integral membrane proteins and perpendicular views over wide areas of the membrane at electron microscopical resolution. This information is directly assessable for 3D analyses and quantitative analyses of the distribution of components within the membrane if it were possible to specifically detect the components of interest in the membranes. Freeze-fracture replica immunolabeling (FRIL) achieves just that. In addition, FRIL preserves antigens in their genuine cellular context free of artifacts of chemical fixation, as FRIL uses chemically unfixed cellular samples that are rapidly cryofixed. In principle, the method is not limited to integral proteins spanning the membrane. Theoretically, all membrane components should be addressable as long as they are antigenic, embedded into at least one membrane leaflet, and accessible for immunolabeling from either the intracellular or the extracellular side. Consistently, integral proteins spanning both leaflets and only partially inserted membrane proteins have been successfully identified and studied for their molecular organization and distribution in the membrane and/or in relationship to specialized membrane domains. Here we describe the freeze-fracturing of both cultured cells and tissues and the sample preparations that allowed for a successful immunogold-labeling of caveolin1 and caveolin3 or even for double-immunolabelings of caveolins with members of the syndapin family of membrane-associating and -shaping BAR domain proteins as well as with cavin 1. For this purpose samples are cryopreserved, fractured, and replicated. We also describe how the obtained stabilized membrane fractures are then cleaned to remove all loosely attached material and immunogold labeled to finally be viewed by transmission electron microscopy.

Key words

Freeze-fracture Membrane topology Immunogold labeling Membrane proteins Membrane-associated proteins Nanodomains Caveolae Caveolar invaginations Caveolin Syndapin 



We would like to acknowledge the support from the Electron Microscopy Center, Jena University Hospital.


  1. 1.
    Steere RL (1957) Electron microscopy of structural detail in frozen biological specimens. J Biophys Biochem Cytol 3(1):45–60CrossRefGoogle Scholar
  2. 2.
    Deamer DW, Branton D (1967) Fracture planes in an ice-bilayer model membrane system. Science 158(3801):655–657CrossRefGoogle Scholar
  3. 3.
    Singer SJ, Nicolson GL (1972) The fluid mosaic model of the structure of cell membranes. Science 175(4023):720–731CrossRefGoogle Scholar
  4. 4.
    Severs NJ (2007) Freeze-fracture electron microscopy. Nat Protoc 2(3):547–576CrossRefGoogle Scholar
  5. 5.
    da Silva PP, Branton D (1970) Membrane splitting in freeze-etching covalently bound ferritin as a membrane marker. J Cell Biol 45(3):598–605CrossRefGoogle Scholar
  6. 6.
    Dinchuk JE, Johnson TJ, Rash JE (1987) Postreplication labeling of E-leaflet molecules: membrane immunoglobulins localized in sectioned, labeled replicas examined by TEM and HVEM. J Electron Microsc Tech 7(1):1–16CrossRefGoogle Scholar
  7. 7.
    Gruijters WT, Kistler J, Bullivant S et al (1987) Immunolocalization of MP70 in lens fiber 16-17-nm intercellular junctions. J Cell Biol 104(3):565–572CrossRefGoogle Scholar
  8. 8.
    Fujimoto K (1995) Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. Application to the immunogold labeling of intercellular junctional complexes. J Cell Sci 108(11):3443–3449PubMedGoogle Scholar
  9. 9.
    Mansouri M, Kasugai Y, Fukazawa Y et al (2015) Distinct subsynaptic localization of type 1 metabotropic glutamate receptors at glutamatergic and GABA ergic synapses in the rodent cerebellar cortex. Eur J Neurosci 41(2):157–167CrossRefGoogle Scholar
  10. 10.
    Kasugai Y, Swinny JD, Roberts JDB et al (2010) Quantitative localisation of synaptic and extrasynaptic GABAA receptor subunits on hippocampal pyramidal cells by freeze-fracture replica immunolabelling. Eur J Neurosci 32(11):1868–1888CrossRefGoogle Scholar
  11. 11.
    Indriati DW, Kamasawa N, Matsui K et al (2013) Quantitative localization of Cav2.1 (P/Q-type) voltage-dependent calcium channels in Purkinje cells: somatodendritic gradient and distinct somatic coclustering with calcium-activated potassium channels. J Neurosci 33(8):3668–3678CrossRefGoogle Scholar
  12. 12.
    Kaufmann W, Kasugai Y, Ferraguti F et al (2010) Two distinct pools of large-conductance calcium-activated potassium channels in the somatic plasma membrane of central principal neurons. Neuroscience 169(3):974–986CrossRefGoogle Scholar
  13. 13.
    Fujimoto T, Kogo H, Nomura R et al (2000) Isoforms of caveolin-1 and caveolar structure. J Cell Sci 113(19):3509–3517PubMedGoogle Scholar
  14. 14.
    Westermann M, Steiniger F, Richter W (2005) Belt-like localisation of caveolin in deep caveolae and its re-distribution after cholesterol depletion. Histochem Cell Biol 123(6):613–620CrossRefGoogle Scholar
  15. 15.
    Koch D, Westermann M, Kessels MM et al (2012) Ultrastructural freeze-fracture immunolabeling identifies plasma membrane-localized syndapin II as a crucial factor in shaping caveolae. Histochem Cell Biol 138(2):215–230CrossRefGoogle Scholar
  16. 16.
    Seemann E, Sun M, Krueger S et al (2017) Deciphering caveolar functions by syndapin III KO-mediated impairment of caveolar invagination. elife 6:e29854CrossRefGoogle Scholar
  17. 17.
    Fujimoto T, Kogo H, Ishiguro K et al (2001) Caveolin-2 is targeted to lipid droplets, a new “membrane domain” in the cell. J Cell Biol 152(5):1079–1085CrossRefGoogle Scholar
  18. 18.
    Schneider K, Seemann E, Liebmann L et al (2014) ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function. J Cell Biol 205(2):197–215CrossRefGoogle Scholar
  19. 19.
    Qualmann B, Roos J, DiGregorio PJ et al (1999) Syndapin I, a synaptic dynamin-binding protein that associates with the neural Wiskott-Aldrich syndrome protein. Mol Biol Cell 10(2):501–513CrossRefGoogle Scholar
  20. 20.
    Qualmann B, Kelly RB (2000) Syndapin isoforms participate in receptor-mediated endocytosis and actin organization. J Cell Biol 148(5):1047–1062CrossRefGoogle Scholar
  21. 21.
    Modregger J, Ritter B, Witter B et al (2000) All three PACSIN isoforms bind to endocytic proteins and inhibit endocytosis. J Cell Sci 113(24):4511–4521PubMedGoogle Scholar
  22. 22.
    Kessels MM, Qualmann B (2004) The syndapin protein family: linking membrane trafficking with the cytoskeleton. J Cell Sci 117(Pt 15):3077–3086CrossRefGoogle Scholar
  23. 23.
    Quan A, Robinson PJ (2013) Syndapin–a membrane remodelling and endocytic F-BAR protein. FEBS J 280(21):5198–5212CrossRefGoogle Scholar
  24. 24.
    Kessels MM, Qualmann B (2015) Different functional modes of BAR domain proteins in formation and plasticity of mammalian postsynapses. J Cell Sci 128(17):3177–3185CrossRefGoogle Scholar
  25. 25.
    McMahon HT, Gallop JL (2005) Membrane curvature and mechanisms of dynamic cell membrane remodelling. Nature 438(7068):590–596CrossRefGoogle Scholar
  26. 26.
    Frost A, Unger VM, De Camilli P (2009) The BAR domain superfamily: membrane-molding macromolecules. Cell 137(2):191–196CrossRefGoogle Scholar
  27. 27.
    Qualmann B, Koch D, Kessels MM (2011) Let’s go bananas: revisiting the endocytic BAR code. EMBO J 30(17):3501–3515CrossRefGoogle Scholar
  28. 28.
    Peter BJ, Kent HM, Mills IG et al (2004) BAR domains as sensors of membrane curvature: the amphiphysin BAR structure. Science 303(5657):495–499CrossRefGoogle Scholar
  29. 29.
    Wang Q, Navarro MV, Peng G et al (2009) Molecular mechanism of membrane constriction and tubulation mediated by the F-BAR protein Pacsin/Syndapin. Proc Natl Acad Sci U S A 106(31):12700–12705CrossRefGoogle Scholar
  30. 30.
    Monier S, Parton RG, Vogel F et al (1995) VIP21-caveolin, a membrane protein constituent of the caveolar coat, oligomerizes in vivo and in vitro. Mol Biol Cell 6(7):911–927CrossRefGoogle Scholar
  31. 31.
    Parton RG, Hanzal-Bayer M, Hancock JF (2006) Biogenesis of caveolae: a structural model for caveolin-induced domain formation. J Cell Sci 119(5):787–796CrossRefGoogle Scholar
  32. 32.
    Zobel T, Brinkmann K, Koch N et al (2015) Cooperative functions of the two F-BAR proteins Cip4 and Nostrin in regulating E-cadherin in epithelial morphogenesis. J Cell Sci 128(3):499–515CrossRefGoogle Scholar
  33. 33.
    Schlörmann W, Steiniger F, Richter W et al (2010) The shape of caveolae is omega-like after glutaraldehyde fixation and cup-like after cryofixation. Histochem Cell Biol 133(2):223–228CrossRefGoogle Scholar
  34. 34.
    Fujita A, Fujimoto T (2007) Quantitative retention of membrane lipids in the freeze-fracture replica. Histochem Cell Biol 128(5):385–389CrossRefGoogle Scholar
  35. 35.
    Schlörmann W, John M, Steiniger F et al (2007) Improved antigen retrieval in freeze-fracture cytochemistry by evaporation of carbon as first replication layer. Histochem Cell Biol 127(6):633–639CrossRefGoogle Scholar
  36. 36.
    Bocker HT, Heinrich T, Liebmann L et al (2019) The Na+/H+ exchanger Nhe1 modulates network excitability via GABA release. Cereb Cortex 29(10):4263–4276CrossRefGoogle Scholar
  37. 37.
    Wolf D, Hofbrucker-MacKenzie SA, Izadi M, Seemann E, Steiniger F, Schwintzer L, Koch D, Kessels MM, Qualmann B (2018) Ankyrin repeat-containing N-Ank proteins shape cellular membranes. Nat Cell Biol 21(10):1191–1205Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Institute for Biochemistry IJena University Hospital – Friedrich Schiller University JenaJenaGermany

Personalised recommendations