Abstract
The ultrastructural analysis of biological membranes by freeze fracture has a 60-year tradition. In this review, we summarize the benefits of the freeze-fracture technique and review special structures analyzed by freeze fracture and by combined freeze-fracture replica immunogold labeling (FRIL) of cell cultures. In principle, every cellular membrane whether of cell suspensions, mono- or bilayers of cell cultures can be analyzed in freeze fracture. The combination of freeze fracture and immunogold labeling of the replica allows the ultrastructural identification of protein assemblies in combination with the molecular identification of their constituent proteins using specific antibodies. The analysis of fractured and labeled intramembrane particles enables determination of the arrangement and organization of proteins within the membrane due to the high resolution of the transmission electron microscope. Because of cell-specific ultrastructural features such as square arrays, identification of cell types can be performed in parallel. This review is aimed at presenting the possibilities of freeze fracture and FRIL in the high-resolution ultrastructural analysis of membrane proteins and their assembly in naïve, transfected or otherwise treated cultured cells. At the interface of molecular approaches and morphology, the application of FRIL in genetically modified cells provides a novel and intriguing aspect for their analysis.
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Abbreviations
- E-face:
-
Extraplasmic leaflet
- FF:
-
Freeze fracture
- FRIL:
-
FF replica immunolabeling
- IMP:
-
Intramembrane particle
- P-face:
-
Protoplasmic leaflet
- Pt/C:
-
Platinum/carbon
- SDS:
-
Sodium dodecyl sulfate
- TEM:
-
Transmission electron microscope
References
Beckmann A, Grissmer A, Krause E, Tschernig T, Meier C (2016a) Pannexin-1 channels show distinct morphology and no gap junction characteristics in mammalian cells. Cell Tissue Res 363(3):751–763. https://doi.org/10.1007/s00441-015-2281-x
Beckmann A, Schubert M, Hainz N, Haase A, Martin U, Tschernig T, Meier C (2016b) Ultrastructural demonstration of Cx43 gap junctions in induced pluripotent stem cells from human cord blood. Histochem Cell Biol 146(5):529–537. https://doi.org/10.1007/s00418-016-1469-9
Boucherie S, Decaens C, Verbavatz JM, Grosse B, Erard M, Merola F, Cassio D, Combettes L (2013) Cadmium disorganises the scaffolding of gap and tight junction proteins in the hepatic cell line WIF B9. Biol Cell 105(12):561–575. https://doi.org/10.1111/boc.201200092
Branton D, Bullivant S, Gilula NB, Karnovsky MJ, Moor H, Muhlethaler K, Northcote DH, Packer L, Satir B, Satir P, Speth V, Staehlin LA, Steere RL, Weinstein RS (1975) Freeze-etching nomenclature. Science 190(4209):54–56
Cheng JP, Nichols BJ (2016) Caveolae: One Function or Many? Trends Cell Biol 26(3):177–189. https://doi.org/10.1016/j.tcb.2015.10.010
De Maziere AM, Scheuermann DW (1990) Structural changes in cardiac gap junctions after hypoxia and reoxygenation: a quantitative freeze-fracture analysis. Cell Tissue Res 261(1):183–194
Dermietzel R (1973) Visualization by freeze-fracturing of regular structures in glial cell membranes. Naturwissenschaften 60(4):208
Dermietzel R (1974) Junctions in the central nervous system of the cat. 3. Gap junctions and membrane-associated orthogonal particle complexes (MOPC) in astrocytic membranes. Cell Tissue Res 149(1):121–135
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):1–16. https://doi.org/10.1002/jemt.1060070102
Dykstra MJ, Reuss LE (2003) Cryotechniques. Biological Electron Microscopy: Theory, Techniques, and Troubleshooting. Springer, pp 125–149
Egbert DD (1950) The Idea of organic Expression and American Architecture. In: Persons S (ed) Evolutionary thought in America. Yale University Press, New Haven
Freese C, Hanada S, Fallier-Becker P, Kirkpatrick CJ, Unger RE (2016) Identification of neuronal and angiogenic growth factors in an in vitro blood-brain barrier model system: relevance in barrier integrity and tight junction formation and complexity. Microvasc Res 111:1–11. https://doi.org/10.1016/j.mvr.2016.12.001
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 (Pt 11):3443–3449
Fujimoto K (1997) SDS-digested freeze-fracture replica labeling electron microscopy to study the two-dimensional distribution of integral membrane proteins and phospholipids in biomembranes: practical procedure, interpretation and application. Histochem Cell Biol 107(2):87–96
Fujita A, Fujimoto T, Ozato-Sakurai N, Suzuki H (2012) A method for efficient observation of intracellular membranes of monolayer culture cells by quick-freeze and freeze-fracture electron microscopy. J Electron Microsc (Tokyo) 61(6):441–446. https://doi.org/10.1093/jmicro/dfs063
Furman CS, Gorelick-Feldman DA, Davidson KG, Yasumura T, Neely JD, Agre P, Rash JE (2003) Aquaporin-4 square array assembly: opposing actions of M1 and M23 isoforms. Proc Natl Acad Sci USA 100(23):13609–13614. https://doi.org/10.1073/pnas.2235843100
Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S (1993) Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol 123(6 Pt 2):1777–1788
Gruijters WT, Kistler J, Bullivant S, Goodenough DA (1987) Immunolocalization of MP70 in lens fiber 16-17-nm intercellular junctions. J Cell Biol 104(3):565–572
Harada H, Shigemoto R (2016) Immunogold protein localization on grid-glued freeze-fracture replicas. Methods Mol Biol (Clifton NJ) 1474:203–216. https://doi.org/10.1007/978-1-4939-6352-2_12
Heuser JE (2011) The origins and evolution of freeze-etch electron microscopy. J Electron Microsc 60 Suppl 1:S3-29. https://doi.org/10.1093/jmicro/dfr044
Heuser JE, Reese TS, Dennis MJ, Jan Y, Jan L, Evans L (1979) Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release. J Cell Biol 81(2):275–300
Hulper P, Veszelka S, Walter FR, Wolburg H, Fallier-Becker P, Piontek J, Blasig IE, Lakomek M, Kugler W, Deli MA (2013) Acute effects of short-chain alkylglycerols on blood-brain barrier properties of cultured brain endothelial cells. Br J Pharmacol 169(7):1561–1573. https://doi.org/10.1111/bph.12218
Inai T, Sengoku A, Hirose E, Iida H, Shibata Y (2009) Freeze-fracture electron microscopic study of tight junction strands in HEK293 cells and MDCK II cells expressing claudin-1 mutants in the second extracellular loop. Histochem Cell Biol 131(6):681–690. https://doi.org/10.1007/s00418-009-0571-7
Indriati DW, Kamasawa N, Matsui K, Meredith AL, Watanabe M, Shigemoto R (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):3668–3678. https://doi.org/10.1523/JNEUROSCI.2921-12.2013
Johnson RG, Reynhout JK, TenBroek EM, Quade BJ, Yasumura T, Davidson KG, Sheridan JD, Rash JE (2012) Gap junction assembly: roles for the formation plaque and regulation by the C-terminus of connexin43. Mol Biol Cell 23 (1):71–86. https://doi.org/10.1091/mbc.E11-02-0141
Jung JS, Bhat RV, Preston GM, Guggino WB, Baraban JM, Agre P (1994) Molecular characterization of an aquaporin cDNA from brain: candidate osmoreceptor and regulator of water balance. Proc Natl Acad Sci USA 91(26):13052–13056
Kaech A, Ziegler U (2014) High-pressure freezing: current state and future prospects. Methods Mol Biol (Clifton NJ) 1117:151–171. https://doi.org/10.1007/978-1-62703-776-1_8
Kan FW (2014) Freeze-fracture immunocytochemistry: fracture-label and label-fracture for the localization of membrane proteins. Curr Protoc Cell Biol 65:4–28. https://doi.org/10.1002/0471143030.cb0428s65
Koch D, Westermann M, Kessels MM, Qualmann B (2012) Ultrastructural freeze-fracture immunolabeling identifies plasma membrane-localized syndapin II as a crucial factor in shaping caveolae. Histochem Cell Biol 138(2):215–230. https://doi.org/10.1007/s00418-012-0945-0
Landis DM, Reese TS (1974) Arrays of particles in freeze-fractured astrocytic membranes. J Cell Biol 60(1):316–320
Li X, Lynn BD, Olson C, Meier C, Davidson KG, Yasumura T, Rash JE, Nagy JI (2002) Connexin29 expression, immunocytochemistry and freeze-fracture replica immunogold labelling (FRIL) in sciatic nerve. Eur J Neurosci 16(5):795–806
Liebner S, Kniesel U, Kalbacher H, Wolburg H (2000) Correlation of tight junction morphology with the expression of tight junction proteins in blood-brain barrier endothelial cells. Eur J Cell Biol 79(10):707–717. https://doi.org/10.1078/0171-9335-00101
Lu M, Lee MD, Smith BL, Jung JS, Agre P, Verdijk MA, Merkx G, Rijss JP, Deen PM (1996) The human AQP4 gene: definition of the locus encoding two water channel polypeptides in brain. Proc Natl Acad Sci USA 93(20):10908–10912
Masugi-Tokita M, Shigemoto R (2007) High-resolution quantitative visualization of glutamate and GABA receptors at central synapses. Curr Opin Neurobiol 17(3):387–393. https://doi.org/10.1016/j.conb.2007.04.012
Meier C, Dermietzel R, Davidson KG, Yasumura T, Rash JE (2004) Connexin32-containing gap junctions in Schwann cells at the internodal zone of partial myelin compaction and in Schmidt–Lanterman incisures. J Neurosci 24(13):3186–3198. https://doi.org/10.1523/JNEUROSCI.5146-03.2004
Milatz S, Piontek J, Hempel C, Meoli L, Grohe C, Fromm A, Lee IM, El-Athman R, Gunzel D (2017) Tight junction strand formation by claudin-10 isoforms and claudin-10a/-10b chimeras. Ann N Y Acad Sci. https://doi.org/10.1111/nyas.13393
Moor H, Muhlethaler K, Waldner H, Frey-Wyssling A (1961) A new freezing-ultramicrotome. J Biophys Biochem Cytol 10:1–13
Nagy JI, Pereda AE, Rash JE (2017) Electrical synapses in mammalian CNS: past eras, present focus and future directions. Biochim Biophys Acta. https://doi.org/10.1016/j.bbamem.2017.05.019
Nassar ZD, Parat MO (2015) Cavin Family: New Players in the Biology of Caveolae. Int Rev Cell Mol Biol 320:235–305. https://doi.org/10.1016/bs.ircmb.2015.07.009
Parajuli LK, Nakajima C, Kulik A, Matsui K, Schneider T, Shigemoto R, Fukazawa Y (2012) Quantitative regional and ultrastructural localization of the Ca(v)2.3 subunit of R-type calcium channel in mouse brain. J Neurosci 32(39):13555–13567. https://doi.org/10.1523/JNEUROSCI.1142-12.2012
Pauli B, Weinstein RS, Soble LW, Alroy J (1977) Freeze-fracture of monolayer cultures. J Cell Biol 72(3):763–769
Peracchia C (1977) Gap junctions. Structural changes after uncoupling procedures. J Cell Biol 72(3):628–641
Pereda A, O’Brien J, Nagy JI, Bukauskas F, Davidson KG, Kamasawa N, Yasumura T, Rash JE (2003) Connexin35 mediates electrical transmission at mixed synapses on Mauthner cells. J Neurosci 23(20):7489–7503
Pfenninger KH, Rinderer ER (1975) Methods for the freeze-fracturing of nerve tissue cultures and cell monolayers. J Cell Biol 65(1):15–28
Phillips TEB, A.F (1984) Liquid nitrogen-based quick freezing: experiences with bounce-free delivery of cholinergic nerve terminals to a metal surface. J Electron Microsc Tech 1(1):9–24
Piontek J, Fritzsche S, Cording J, Richter S, Hartwig J, Walter M, Yu D, Turner JR, Gehring C, Rahn HP, Wolburg H, Blasig IE (2011) Elucidating the principles of the molecular organization of heteropolymeric tight junction strands. Cellular Mol Life Sci 68(23):3903–3918. https://doi.org/10.1007/s00018-011-0680-z
Piontek A, Rossa J, Protze J, Wolburg H, Hempel C, Gunzel D, Krause G, Piontek J (2017) Polar and charged extracellular residues conserved among barrier-forming claudins contribute to tight junction strand formation. Ann N Y Acad Sci 1397(1):143–156. https://doi.org/10.1111/nyas.13341
Rash JE (1983) The rapid-freeze technique in neurobiology. Trends Neurosci 6:208–212
Rash JE, Ellisman MH (1974) Studies of excitable membranes. I. Macromolecular specializations of the neuromuscular junction and the nonjunctional sarcolemma. J Cell Biol 63(2 Pt 1):567–586
Rash JE, Giddings FD (1989) Counting and measuring IMPs and pits: why accurate counts are exceedingly rare. J Electron Microsc Tech 13(3):204–215. https://doi.org/10.1002/jemt.1060130307
Rash JE, Yasumura T (1992) Improved structural detail in freeze-fracture replicas: high-angle shadowing of gap junctions cooled below – 170 degrees C and protected by liquid nitrogen-cooled shrouds. Microsc Res Tech 20(2):187–204. https://doi.org/10.1002/jemt.1070200207
Rash JE, Yasumura T (1999) Direct immunogold labeling of connexins and aquaporin-4 in freeze-fracture replicas of liver, brain, and spinal cord: factors limiting quantitative analysis. Cell Tissue Res 296(2):307–321
Rash JE, Staehelin LA, Ellisman MH (1974) Rectangular arrays of particles on freeze-cleaved plasma membranes are not gap junctions. Exp Cell Res 86(1):187–190
Rash JE, Duffy HS, Dudek FE, Bilhartz BL, Whalen LR, Yasumura T (1997) Grid-mapped freeze-fracture analysis of gap junctions in gray and white matter of adult rat central nervous system, with evidence for a “panglial syncytium” that is not coupled to neurons. J Comp Neurol 388(2):265–292
Rash JE, Yasumura T, Hudson CS, Agre P, Nielsen S (1998) Direct immunogold labeling of aquaporin-4 in square arrays of astrocyte and ependymocyte plasma membranes in rat brain and spinal cord. Proc Natl Acad Sci USA 95(20):11981–11986
Rash JE, Pereda A, Kamasawa N, Furman CS, Yasumura T, Davidson KG, Dudek FE, Olson C, Li X, Nagy JI (2004) High-resolution proteomic mapping in the vertebrate central nervous system: close proximity of connexin35 to NMDA glutamate receptor clusters and co-localization of connexin36 with immunoreactivity for zonula occludens protein-1 (ZO-1). J Neurocytol 33(1):131–151. https://doi.org/10.1023/B:NEUR.0000029653.34094.0b
Rash JE, Davidson KG, Kamasawa N, Yasumura T, Kamasawa M, Zhang C, Michaels R, Restrepo D, Ottersen OP, Olson CO, Nagy JI (2005) Ultrastructural localization of connexins (Cx36, Cx43, Cx45), glutamate receptors and aquaporin-4 in rodent olfactory mucosa, olfactory nerve and olfactory bulb. J Neurocytol 34(3–5):307–341. https://doi.org/10.1007/s11068-005-8360-2
Rash JE, Kamasawa N, Davidson KG, Yasumura T, Pereda AE, Nagy JI (2012) Connexin composition in apposed gap junction hemiplaques revealed by matched double-replica freeze-fracture replica immunogold labeling. J Membr Biol 245(5–6):333–344. https://doi.org/10.1007/s00232-012-9454-2
Rash JE, Vanderpool KG, Yasumura T, Hickman J, Beatty JT, Nagy JI (2016) KV1 channels identified in rodent myelinated axons, linked to Cx29 in innermost myelin: support for electrically active myelin in mammalian saltatory conduction. J Neurophysiol 115(4):1836–1859. https://doi.org/10.1152/jn.01077.2015
Robenek H, Severs NJ (2008) Recent advances in freeze-fracture electron microscopy: the replica immunolabeling technique. Biol Proced Online 10:9–19. https://doi.org/10.1251/bpo138
Robenek H, Weissen-Plenz G, Severs NJ (2008) Freeze-fracture replica immunolabelling reveals caveolin-1 in the human cardiomyocyte plasma membrane. J Cell Mol Med 12(6A):2519–2521. https://doi.org/10.1111/j.1582-4934.2008.00498.x
Robenek H, Buers I, Hofnagel O, Lorkowski S, Severs NJ (2009a) GFP-tagged proteins visualized by freeze-fracture immuno-electron microscopy: a new tool in cellular and molecular medicine. J Cell Mol Med 13(7):1381–1390. https://doi.org/10.1111/j.1582-4934.2008.00407.x
Robenek H, Buers I, Hofnagel O, Robenek MJ, Troyer D, Severs NJ (2009b) Compartmentalization of proteins in lipid droplet biogenesis. Biochim Biophys Acta 1791(6):408–418. https://doi.org/10.1016/j.bbalip.2008.12.001
Rosenthal R, Luettig J, Hering NA, Krug SM, Albrecht U, Fromm M, Schulzke JD (2016) Myrrh exerts barrier-stabilising and -protective effects in HT-29/B6 and Caco-2 intestinal epithelial cells. Int J Colorectal Dis. https://doi.org/10.1007/s00384-016-2736-x
Rossa J, Ploeger C, Vorreiter F, Saleh T, Protze J, Gunzel D, Wolburg H, Krause G, Piontek J (2014) Claudin-3 and claudin-5 protein folding and assembly into the tight junction are controlled by non-conserved residues in the transmembrane 3 (TM3) and extracellular loop 2 (ECL2) segments. J Biol Chem 289(11):7641–7653. https://doi.org/10.1074/jbc.M113.531012
Schlormann W, Steiniger F, Richter W, Kaufmann R, Hause G, Lemke C, Westermann M (2010) The shape of caveolae is omega-like after glutaraldehyde fixation and cup-like after cryofixation. Histochem Cell Biol 133(2):223–228. https://doi.org/10.1007/s00418-009-0651-8
Sengoku A, Inai T, Shibata Y (2008) Formation of aberrant TJ strands by overexpression of claudin-15 in MDCK II cells. Histochem Cell Biol 129(2):211–222. https://doi.org/10.1007/s00418-007-0354-y
Severs NJ (2000) The cardiac muscle cell. Bioessays 22 (2):188–199. https://doi.org/10.1002/(SICI)1521-1878(200002)22:2%3C188::AID-BIES10%3E3.0.CO;2-T
Severs NJ (2007) Freeze-fracture electron microscopy. Nat Protoc 2(3):547–576. https://doi.org/10.1038/nprot.2007.55
Severs NJ, Robenek H (2008) Freeze-fracture cytochemistry in cell biology. Methods Cell Biol 88:181–204. https://doi.org/10.1016/s0091-679x(08)00411-1
Staehelin LA, Mukherjee TM, Williams AW (1969) Freeze-etch appearance of the tight junctions in the epithelium of small and large intestine of mice. Protoplasma 67(2):165–184
Steere RL (1957) Electron microscopy of structural detail in frozen biological specimens. J Biophys Biochem Cytol 3(1):45–60
Steere RL (1969) Freeze-etching and direct observation of freezing damage. Cryobiology 6(3):137–150
Steere RL (1983) Supporting freeze-etch specimens with “Lexan” while dissolving biological remains in acid. Proc EMSA 41:618–619
Todd JH, Sens DA, Sens MA, Hazen-Martin D (1992) In situ freeze-fracture of monolayer cell cultures grown on a permeable support. Microsc Res Tech 22(3):301–305. https://doi.org/10.1002/jemt.1070220308
Umrath W (1981) An improved technique for the freeze-fracture of cell cultures grown as monolayers. J Microsc 121(Pt 2):229–234
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):613–620. https://doi.org/10.1007/s00418-004-0750-5
Wolburg H, Neuhaus J, Kniesel U, Krauss B, Schmid EM, Ocalan M, Farrell C, Risau W (1994) Modulation of tight junction structure in blood-brain barrier endothelial cells. Effects of tissue culture, second messengers and cocultured astrocytes. J Cell Sci 107 (Pt 5):1347–1357
Wolburg-Buchholz K, Mack AF, Steiner E, Pfeiffer F, Engelhardt B, Wolburg H (2009) Loss of astrocyte polarity marks blood-brain barrier impairment during experimental autoimmune encephalomyelitis. Acta Neuropathol (Berl) 118(2):219–233. https://doi.org/10.1007/s00401-009-0558-4
Acknowledgements
The authors wish to thank John E. Rash for his expert advice and continuous support. Thanks also to Alexander Grissmer for excellent technical support and to Franziska Müller and Alina Mattheis for the expertly drawn illustration. The authors acknowledge financial support by the German Research Foundation and the Saarland, who funded the freeze-fracture unit.
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Figure 3a has been reproduced from the Springer article “SDS-digested freeze-fracture replica labeling electron microscopy to study the two-dimensional distribution of integral membrane proteins and phospholipids in biomembranes: practical procedure, interpretation and application” by Kazushi Fujimoto (1997) Histochemistry and Cell Biology 107: 87–96. With permission of Springer.
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Meier, C., Beckmann, A. Freeze fracture: new avenues for the ultrastructural analysis of cells in vitro. Histochem Cell Biol 149, 3–13 (2018). https://doi.org/10.1007/s00418-017-1617-x
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DOI: https://doi.org/10.1007/s00418-017-1617-x