freeze fracturing


Summary: Preparation for electron microscopy of minute replicas of exposed surfaces of the cell which have been ruptured in the frozen state. The specimen is frozen, then cleaved under high vacuum at the same temperature. The exposed surface is shadowed with carbon and platinum and coated with carbon to obtain a carbon replica.

Top Publications

  1. Fallier Becker P, Sperveslage J, Wolburg H, Noell S. The impact of agrin on the formation of orthogonal arrays of particles in cultured astrocytes from wild-type and agrin-null mice. Brain Res. 2011;1367:2-12 pubmed publisher
  2. Biswas S, Lo W. Gap junctions contain different amounts of cholesterol which undergo unique sequestering processes during fiber cell differentiation in the embryonic chicken lens. Mol Vis. 2007;13:345-59 pubmed
  3. Rash J, Olson C, Pouliot W, Davidson K, Yasumura T, Furman C, et al. Connexin36 vs. connexin32, "miniature" neuronal gap junctions, and limited electrotonic coupling in rodent suprachiasmatic nucleus. Neuroscience. 2007;149:350-71 pubmed
  4. Robenek H, Buers I, Hofnagel O, Lorkowski S, Severs N. GFP-tagged proteins visualized by freeze-fracture immuno-electron microscopy: a new tool in cellular and molecular medicine. J Cell Mol Med. 2009;13:1381-90 pubmed publisher
    ..The application of this approach is illustrated by new findings on PAT-family proteins tagged with GFP transfected into fibroblasts from patients with Niemann-Pick type C disease. ..
  5. Severs N, Robenek H. Freeze-fracture cytochemistry in cell biology. Methods Cell Biol. 2008;88:181-204 pubmed publisher
    ..Examples of how these techniques have contributed to our understanding of cardiovascular cell function in health and disease are discussed. ..
  6. Zampighi G, Planells A, Lin D, Takemoto D. Regulation of lens cell-to-cell communication by activation of PKCgamma and disassembly of Cx50 channels. Invest Ophthalmol Vis Sci. 2005;46:3247-55 pubmed
    ..The findings also suggest that Cx50 channel disassembly occurs in distinct lipid microdomains. ..
  7. Kamasawa N, Furman C, Davidson K, Sampson J, Magnie A, Gebhardt B, et al. Abundance and ultrastructural diversity of neuronal gap junctions in the OFF and ON sublaminae of the inner plexiform layer of rat and mouse retina. Neuroscience. 2006;142:1093-117 pubmed
    ..ON sublamina. ..
  8. Fujita A, Cheng J, Hirakawa M, Furukawa K, Kusunoki S, Fujimoto T. Gangliosides GM1 and GM3 in the living cell membrane form clusters susceptible to cholesterol depletion and chilling. Mol Biol Cell. 2007;18:2112-22 pubmed
    ..The present method enabled to capture the molecular distribution of lipids in the cell membrane, and demonstrated that GM1 and GM3 form clusters that are susceptible to cholesterol depletion and chilling. ..
  9. Fujita A, Fujimoto T. Quantitative retention of membrane lipids in the freeze-fracture replica. Histochem Cell Biol. 2007;128:385-9 pubmed
    ..These results suggest that probes can bind to lipids captured by carbon more efficiently than those captured by platinum. Nonetheless, evaporation of platinum after carbon is indispensable for proper labeling. ..

More Information


  1. Noell S, Fallier Becker P, Beyer C, Kroger S, Mack A, Wolburg H. Effects of agrin on the expression and distribution of the water channel protein aquaporin-4 and volume regulation in cultured astrocytes. Eur J Neurosci. 2007;26:2109-18 pubmed
    ..Implications for the regulation and maintenance of the blood-brain barrier including oedema formation under pathological conditions are discussed. ..
  2. Zampighi G. Distribution of connexin50 channels and hemichannels in lens fibers: a structural approach. Cell Commun Adhes. 2003;10:265-70 pubmed
    ..Therefore, in lens fibers, Cx50 hemichannels are inserted via exocytosis and are rapidly assembled into channels assembled in gap junction plaques. ..
  3. Fujita A, Cheng J, Fujimoto T. Quantitative electron microscopy for the nanoscale analysis of membrane lipid distribution. Nat Protoc. 2010;5:661-9 pubmed publisher
    ..A major advantage of this method is that it does not require the expression of artificial probes. Therefore, this method can be applied to any cell in vitro or in vivo, and the whole procedure can be completed in 1-2 d. ..
  4. Zampighi G, Eskandari S, Hall J, Zampighi L, Kreman M. Micro-domains of AQP0 in lens equatorial fibers. Exp Eye Res. 2002;75:505-19 pubmed
    ..We concluded that the ability of AQP0 to arrange itself in micro-domains conferred functional properties that might contribute to the maintenance of lens transparency and homeostasis. ..
  5. Rash J, Olson C, Davidson K, Yasumura T, Kamasawa N, Nagy J. Identification of connexin36 in gap junctions between neurons in rodent locus coeruleus. Neuroscience. 2007;147:938-56 pubmed
  6. Paolini C, Fessenden J, Pessah I, Franzini Armstrong C. Evidence for conformational coupling between two calcium channels. Proc Natl Acad Sci U S A. 2004;101:12748-52 pubmed
    ..We find a substantial ( approximately 2-nm) shift in the alpha(1S)DHPR positions, indicating that ryanodine induces large conformational changes in the RyR1 cytoplasmic domain and that the alpha(1S)DHPR-RyR complex acts as a unit. ..
  7. Silberstein C, Bouley R, Huang Y, Fang P, Pastor Soler N, Brown D, et al. Membrane organization and function of M1 and M23 isoforms of aquaporin-4 in epithelial cells. Am J Physiol Renal Physiol. 2004;287:F501-11 pubmed
  8. Moore E, Voigt T, Kobayashi Y, Isenberg G, Fay F, Gallitelli M, et al. Organization of Ca2+ release units in excitable smooth muscle of the guinea-pig urinary bladder. Biophys J. 2004;87:1836-47 pubmed
    ..Structural analogies between smooth and cardiac muscle excitation-contraction coupling complexes suggest a common basic mechanism of action. ..
  9. Rash J, Davidson K, Yasumura T, Furman C. Freeze-fracture and immunogold analysis of aquaporin-4 (AQP4) square arrays, with models of AQP4 lattice assembly. Neuroscience. 2004;129:915-34 pubmed
    ..Several structural models are considered that incorporate freeze-fracture data for submolecular "cross-bridges" linking IMPs into the classical square lattices that characterize, in particular, naturally occurring AQP4. ..
  10. Takekura H, Franzini Armstrong C. The structure of Ca(2+) release units in arthropod body muscle indicates an indirect mechanism for excitation-contraction coupling. Biophys J. 2002;83:2742-53 pubmed
    ..This matches the architecture of vertebrate cardiac muscle and is in keeping with the similarity in e-c coupling mechanisms in cardiac and invertebrate striated muscles. ..
  11. Rash J, Davidson K, Kamasawa N, Yasumura T, Kamasawa M, Zhang C, et al. Ultrastructural localization of connexins (Cx36, Cx43, Cx45), glutamate receptors and aquaporin-4 in rodent olfactory mucosa, olfactory nerve and olfactory bulb. J Neurocytol. 2005;34:307-41 pubmed
  12. Protasi F, Paolini C, Nakai J, Beam K, Franzini Armstrong C, Allen P. Multiple regions of RyR1 mediate functional and structural interactions with alpha(1S)-dihydropyridine receptors in skeletal muscle. Biophys J. 2002;83:3230-44 pubmed
  13. Li X, Kamasawa N, Ciolofan C, Olson C, Lu S, Davidson K, et al. Connexin45-containing neuronal gap junctions in rodent retina also contain connexin36 in both apposing hemiplaques, forming bihomotypic gap junctions, with scaffolding contributed by zonula occludens-1. J Neurosci. 2008;28:9769-89 pubmed publisher
    ..These data document that in Cx45-expressing neurons of IPL, Cx45 is almost always accompanied by Cx36, forming "bihomotypic" gap junctions, with Cx45 structurally coupling to Cx45 and Cx36 coupling to Cx36. ..
  14. Noell S, Fallier Becker P, Deutsch U, Mack A, Wolburg H. Agrin defines polarized distribution of orthogonal arrays of particles in astrocytes. Cell Tissue Res. 2009;337:185-95 pubmed publisher
    ..These results clearly demonstrate, for the first time, that agrin plays a pivotal role for the clustering of OAPs in the endfoot membranes of astrocytes, whereas the mere presence of AQP4 is not sufficient for OAP clustering. ..
  15. Biswas S, Jiang J, Lo W. Gap junction remodeling associated with cholesterol redistribution during fiber cell maturation in the adult chicken lens. Mol Vis. 2009;15:1492-508 pubmed
    ..As a result, it compensates considerably for the large decrease in the percentage of membrane area specialized as gap junctions in the mature inner fibers in the adult chicken lens. ..
  16. Fontana J, Lopez Iglesias C, Tzeng W, Frey T, Fernández J, Risco C. Three-dimensional structure of Rubella virus factories. Virology. 2010;405:579-91 pubmed publisher
    ..Immunogold labelling confirmed that the mitochondrial protein p32 is an abundant component around and inside CPVs where it could play important roles in factory activities. ..
  17. Wolburg H, Wolburg Buchholz K, Fallier Becker P, Noell S, Mack A. Structure and functions of aquaporin-4-based orthogonal arrays of particles. Int Rev Cell Mol Biol. 2011;287:1-41 pubmed publisher
    ..In particular, astrocytes and glioma cells will play the major part in this review, not only due to our own work but also due to the fact that most studies on structure and function of AQP4 were done in the nervous system. ..
  18. Furman C, Gorelick Feldman D, Davidson K, Yasumura T, Neely J, Agre P, et al. Aquaporin-4 square array assembly: opposing actions of M1 and M23 isoforms. Proc Natl Acad Sci U S A. 2003;100:13609-14 pubmed
    ..These studies show that M23 and M1 isoforms have opposing effects on intramembrane organization of AQP4: M23 forms large square arrays with abundant cross-bridges; M1 restricts square array assembly. ..
  19. Takekura H, Paolini C, Franzini Armstrong C, Kugler G, Grabner M, Flucher B. Differential contribution of skeletal and cardiac II-III loop sequences to the assembly of dihydropyridine-receptor arrays in skeletal muscle. Mol Biol Cell. 2004;15:5408-19 pubmed
    ..These findings suggest an inhibitory role in tetrad formation of the cardiac II-III loop and that the organization of DHPRs in tetrads vis-a-vis the RyR is necessary but not sufficient for skeletal-type e-c coupling. ..
  20. Moroi S, Saitou M, Fujimoto K, Sakakibara A, Furuse M, Yoshida O, et al. Occludin is concentrated at tight junctions of mouse/rat but not human/guinea pig Sertoli cells in testes. Am J Physiol. 1998;274:C1708-17 pubmed
  21. Furuse M, Sasaki H, Tsukita S. Manner of interaction of heterogeneous claudin species within and between tight junction strands. J Cell Biol. 1999;147:891-903 pubmed
    ..We concluded that distinct species of claudins can interact within and between TJ strands, except in some combinations. This mode of assembly of claudins could increase the diversity of the structure and functions of TJ strands. ..
  22. Hirase T, Staddon J, Saitou M, Ando Akatsuka Y, Itoh M, Furuse M, et al. Occludin as a possible determinant of tight junction permeability in endothelial cells. J Cell Sci. 1997;110 ( Pt 14):1603-13 pubmed
    ..Our data indicate that regulation of occludin expression may be a crucial determinant of the tight junction permeability properties of endothelial cells in different tissues. ..
  23. Nagy J, Li X, Rempel J, Stelmack G, Patel D, Staines W, et al. Connexin26 in adult rodent central nervous system: demonstration at astrocytic gap junctions and colocalization with connexin30 and connexin43. J Comp Neurol. 2001;441:302-23 pubmed
  24. Fujimoto K, Nagafuchi A, Tsukita S, Kuraoka A, Ohokuma A, Shibata Y. Dynamics of connexins, E-cadherin and alpha-catenin on cell membranes during gap junction formation. J Cell Sci. 1997;110 ( Pt 3):311-22 pubmed
    ..In addition, connexin-immunoreactivity was also observed along tight junctional strands, suggesting that the gap junction may also form along the tight junctions. ..
  25. Protasi F, Franzini Armstrong C, Flucher B. Coordinated incorporation of skeletal muscle dihydropyridine receptors and ryanodine receptors in peripheral couplings of BC3H1 cells. J Cell Biol. 1997;137:859-70 pubmed
  26. Meyer H, Richter W. Freeze-fracture studies on lipids and membranes. Micron. 2001;32:615-44 pubmed
  27. Vanmarle J, Vrensen G. Cholesterol content of focal opacities and multilamellar bodies in the human lens: filipin cytochemistry and freeze fracture. Ophthalmic Res. 2000;32:285-91 pubmed
  28. Saitou M, Fujimoto K, Doi Y, Itoh M, Fujimoto T, Furuse M, et al. Occludin-deficient embryonic stem cells can differentiate into polarized epithelial cells bearing tight junctions. J Cell Biol. 1998;141:397-408 pubmed
    ..These findings indicate that there are as yet unidentified TJ integral membrane protein(s) which can form strand structures, recruit ZO-1, and function as a barrier without occludin. ..
  29. Rash J, Yasumura T, Hudson C, Agre P, Nielsen S. 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 U S A. 1998;95:11981-6 pubmed
  30. Gow A, Southwood C, Li J, Pariali M, Riordan G, Brodie S, et al. CNS myelin and sertoli cell tight junction strands are absent in Osp/claudin-11 null mice. Cell. 1999;99:649-59 pubmed
    ..These novel results provide direct evidence of the pivotal role of the claudin family in generating the paracellular physical barrier of tight junctions necessary for spermatogenesis and normal CNS function. ..
  31. Saitou M, Ando Akatsuka Y, Itoh M, Furuse M, Inazawa J, Fujimoto K, et al. Mammalian occludin in epithelial cells: its expression and subcellular distribution. Eur J Cell Biol. 1997;73:222-31 pubmed
    ..Furthermore, the exclusive concentration of occludin at tight junctions in epithelial cells was confirmed by immunoreplica electron microscopy. ..
  32. Fujimoto K. 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. 1997;107:87-96 pubmed
    ..In this review, we describe the practical procedure for SDS-FRL in detail, present its application to labeling of various membrane components, and briefly discuss the possibility of a combination of SDS-FRL with atomic force microscopy. ..
  33. Schneeberger E, Lynch R. Structure, function, and regulation of cellular tight junctions. Am J Physiol. 1992;262:L647-61 pubmed
    ..However, until the biochemical composition of this structure has been defined and its gene identified, the TJ will continue to be an elusive yet tantalizing challenge to the cell biologist. ..
  34. Nelles E, Bützler C, Jung D, Temme A, Gabriel H, Dahl U, et al. Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin32-deficient mice. Proc Natl Acad Sci U S A. 1996;93:9565-70 pubmed
    ..It is possible, however, that they may develop neurodegenerative symptoms at older age. ..
  35. Takekura H, Nishi M, Noda T, Takeshima H, Franzini Armstrong C. Abnormal junctions between surface membrane and sarcoplasmic reticulum in skeletal muscle with a mutation targeted to the ryanodine receptor. Proc Natl Acad Sci U S A. 1995;92:3381-5 pubmed
    ..Since junctions form in the absence of feet and tetrads, coupling of SR to surface membrane and T tubules appears to be mediated by additional proteins, distinct from either RyRs or DHPRs. ..
  36. Rash J, Yasumura T, Dudek F, Nagy J. Cell-specific expression of connexins and evidence of restricted gap junctional coupling between glial cells and between neurons. J Neurosci. 2001;21:1983-2000 pubmed
    ..Thus, the different cell types of the CNS express different connexins, which define separate pathways for neuronal versus glial gap junctional communication. ..
  37. van Hoek A, Ma T, Yang B, Verkman A, Brown D. Aquaporin-4 is expressed in basolateral membranes of proximal tubule S3 segments in mouse kidney. Am J Physiol Renal Physiol. 2000;278:F310-6 pubmed
    ..The functional significance of the apparent species-dependent expression of AQP4 in proximal tubules is unknown, but may relate to physiological differences between rats and mice...
  38. Felder E, Franzini Armstrong C. Type 3 ryanodine receptors of skeletal muscle are segregated in a parajunctional position. Proc Natl Acad Sci U S A. 2002;99:1695-700 pubmed
    ..On the basis of these two observations, we postulate that RyR3s are restricted to the parajunctional region, and thus their activation must be indirect and derivative during excitation-contraction coupling...
  39. Johnson R, Meyer R, Li X, Preus D, Tan L, Grunenwald H, et al. Gap junctions assemble in the presence of cytoskeletal inhibitors, but enhanced assembly requires microtubules. Exp Cell Res. 2002;275:67-80 pubmed
    ..However, microtubules are necessary for enhanced GJ growth and likely for facilitating connexin trafficking under basal conditions. ..
  40. Rash J, Yasumura T. 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. 1999;296:307-21 pubmed
  41. Tsukita S, Furuse M. Occludin and claudins in tight-junction strands: leading or supporting players?. Trends Cell Biol. 1999;9:268-73 pubmed
    ..This review discusses current understanding of the molecular architecture of tight-junction strands, focusing on the recent discovery of two distinct types of tight-junction-specific integral membrane proteins, occludin and claudins. ..
  42. Yang B, Brown D, Verkman A. The mercurial insensitive water channel (AQP-4) forms orthogonal arrays in stably transfected Chinese hamster ovary cells. J Biol Chem. 1996;271:4577-80 pubmed
    ..These results provide direct evidence that a molecular water channel can spontaneously assemble in regular arrays. ..
  43. Morita K, Sasaki H, Fujimoto K, Furuse M, Tsukita S. Claudin-11/OSP-based tight junctions of myelin sheaths in brain and Sertoli cells in testis. J Cell Biol. 1999;145:579-88 pubmed
  44. Furuse M, Sasaki H, Fujimoto K, Tsukita S. A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts. J Cell Biol. 1998;143:391-401 pubmed
    ..These findings suggested that claudin-1 and -2 are mainly responsible for TJ strand formation, and that occludin is an accessory protein in some function of TJ strands. ..
  45. Kamasawa N, Sik A, Morita M, Yasumura T, Davidson K, Nagy J, et al. Connexin-47 and connexin-32 in gap junctions of oligodendrocyte somata, myelin sheaths, paranodal loops and Schmidt-Lanterman incisures: implications for ionic homeostasis and potassium siphoning. Neuroscience. 2005;136:65-86 pubmed
    ..Acting in series and in parallel, autologous and heterologous oligodendrocyte gap junctions provide essential pathways for intra- and intercellular ionic homeostasis. ..
  46. Plattner H. My favorite cell--Paramecium. Bioessays. 2002;24:649-58 pubmed
    ..Although a variety of basic cellular functions are briefly addressed to demonstrate the uniqueness of this unicellular organism, this article focuses on exocytosis regulation...
  47. Hagiwara A, Fukazawa Y, Deguchi Tawarada M, Ohtsuka T, Shigemoto R. Differential distribution of release-related proteins in the hippocampal CA3 area as revealed by freeze-fracture replica labeling. J Comp Neurol. 2005;489:195-216 pubmed
    ..These results support the involvement of the t-SNARE proteins in exocytotic fusion in the AZ and the role of CAST in specialization of the membrane domain for the AZ. ..
  48. Imura T, Yanagishita H, Kitamoto D. Coacervate formation from natural glycolipid: one acetyl group on the headgroup triggers coacervate-to-vesicle transition. J Am Chem Soc. 2004;126:10804-5 pubmed
  49. Westerhuis W, Sturgis J, Ratcliffe E, Hunter C, Niederman R. Isolation, size estimates, and spectral heterogeneity of an oligomeric series of light-harvesting 1 complexes from Rhodobacter sphaeroides. Biochemistry. 2002;41:8698-707 pubmed
  50. Honeywell Nguyen P, de Graaff A, Groenink H, Bouwstra J. The in vivo and in vitro interactions of elastic and rigid vesicles with human skin. Biochim Biophys Acta. 2002;1573:130-40 pubmed
    ..Elastic vesicles and micelles demonstrated very different interactions with human skin and hence probably also have different mechanisms of action for the enhancement of drug transport. ..
  51. Melilli C, Carcò D, Barbano D, Tumino G, Carpino S, Licitra G. Composition, microstructure, and surface barrier layer development during brine salting. J Dairy Sci. 2005;88:2329-40 pubmed
    ..9% at 4 d. There appears to be some critical concentration of salt in brine above which there is a large negative impact on salt uptake due to the creation of a barrier layer at the surface of the block of cheese. ..
  52. Rahner C, Fukuhara M, Peng S, Kojima S, Rizzolo L. The apical and basal environments of the retinal pigment epithelium regulate the maturation of tight junctions during development. J Cell Sci. 2004;117:3307-18 pubmed
    ..Therefore, this experimental model can isolate the effects of retinal secretions on structure and claudin expression, and can help us to determine how claudins affect function when structure is held constant. ..
  53. Zhigaltsev I, Maurer N, Wong K, Cullis P. Triggered release of doxorubicin following mixing of cationic and anionic liposomes. Biochim Biophys Acta. 2002;1565:129-35 pubmed
    ..It is concluded that drug release triggered by mixing anionic and cationic liposomes could be of utility in drug delivery applications. ..