Recent Advances in Freeze-fracture Electron Microscopy: the Replica Immunolabeling Technique

Overview

Journal Biol Proced Online

Publisher Biomed Central

Specialty Biomedical Engineering

Date 2008 Apr 4

PMID 18385807

Citations 10

Authors

Horst Robenek

Nicholas J Severs

Affiliations

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Abstract

Freeze-fracture electron microscopy is a technique for examining the ultrastructure of rapidly frozen biological samples by transmission electron microscopy. Of a range of approaches to freeze-fracture cytochemistry that have been developed and tried, the most successful is the technique termed freeze-fracture replica immunogold labeling (FRIL). In this technique, samples are frozen, fractured and replicated with platinum-carbon as in standard freeze fracture, and then carefully treated with sodium dodecylsulphate to remove all the biological material except a fine layer of molecules attached to the replica itself. Immunogold labeling of these molecules permits their distribution to be seen superimposed upon high resolution planar views of membrane structure. Examples of how this technique has contributed to our understanding of lipid droplet biogenesis and function are discussed.

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References

Robenek M, Severs N, Schlattmann K, Plenz G, Zimmer K, Troyer D . Lipids partition caveolin-1 from ER membranes into lipid droplets: updating the model of lipid droplet biogenesis. FASEB J. 2004; 18(7):866-8. DOI: 10.1096/fj.03-0782fje. View

Rash J, Davidson K, Kamasawa N, Yasumura T, Kamasawa M, Zhang C . Ultrastructural localization of connexins (Cx36, Cx43, Cx45), glutamate receptors and aquaporin-4 in rodent olfactory mucosa, olfactory nerve and olfactory bulb. J Neurocytol. 2006; 34(3-5):307-41. PMC: 1525003. DOI: 10.1007/s11068-005-8360-2. View

Forsman C, Pinto da Silva P . Label-fracture of cell surfaces by replica staining. J Histochem Cytochem. 1988; 36(11):1413-8. DOI: 10.1177/36.11.2459187. View

Robenek H, Lorkowski S, Schnoor M, Troyer D . Spatial integration of TIP47 and adipophilin in macrophage lipid bodies. J Biol Chem. 2004; 280(7):5789-94. DOI: 10.1074/jbc.M407194200. View

Nagy J, Dudek F, Rash J . Update on connexins and gap junctions in neurons and glia in the mammalian nervous system. Brain Res Brain Res Rev. 2004; 47(1-3):191-215. DOI: 10.1016/j.brainresrev.2004.05.005. View

Robenek H, Hofnagel O, Buers I, Robenek M, Troyer D, Severs N . Adipophilin-enriched domains in the ER membrane are sites of lipid droplet biogenesis. J Cell Sci. 2006; 119(Pt 20):4215-24. DOI: 10.1242/jcs.03191. View

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(2):87-96. DOI: 10.1007/s004180050092. View

Kamasawa N, Sik A, Morita M, Yasumura T, Davidson K, Nagy J . 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(1):65-86. PMC: 1550704. DOI: 10.1016/j.neuroscience.2005.08.027. View

Branton D, Bullivant S, Gilula N, Karnovsky M, Moor H, Muhlethaler K . Freeze-etching nomenclature. Science. 1975; 190(4209):54-6. DOI: 10.1126/science.1166299. View

10.

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(20):11981-6. PMC: 21751. DOI: 10.1073/pnas.95.20.11981. View

11.

Robenek M, Schlattmann K, Zimmer K, Plenz G, Troyer D, Robenek H . Cholesterol transporter caveolin-1 transits the lipid bilayer during intracellular cycling. FASEB J. 2003; 17(13):1940-2. DOI: 10.1096/fj.03-0008fje. View

12.

Fujimoto K . 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. 1995; 108 ( Pt 11):3443-9. DOI: 10.1242/jcs.108.11.3443. View

13.

Stevenson S, Rothery S, Cullen M, Severs N . Spatial relationship of the C-terminal domains of dystrophin and beta-dystroglycan in cardiac muscle support a direct molecular interaction at the plasma membrane interface. Circ Res. 1998; 82(1):82-93. DOI: 10.1161/01.res.82.1.82. View

14.

Da Silva P, Forsman C, Fujimoto K . Fracture-flip: nanoanatomy and topochemistry of cell surfaces. Prog Clin Biol Res. 1989; 295:49-56. View

15.

Kamasawa N, Furman C, Davidson K, Sampson J, Magnie A, Gebhardt B . 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(4):1093-117. PMC: 1847771. DOI: 10.1016/j.neuroscience.2006.08.020. View

16.

Pinto da Silva P, Kan F . Label-fracture: a method for high resolution labeling of cell surfaces. J Cell Biol. 1984; 99(3):1156-61. PMC: 2113382. DOI: 10.1083/jcb.99.3.1156. View

17.

Dunia I, Recouvreur M, Nicolas P, Kumar N, Bloemendal H, Benedetti E . Sodium dodecyl sulfate-freeze-fracture immunolabeling of gap junctions. Methods Mol Biol. 2001; 154:33-55. DOI: 10.1385/1-59259-043-8:33. View

18.

Stevenson S, Cullen M, Rothery S, Coppen S, Severs N . High-resolution en-face visualization of the cardiomyocyte plasma membrane reveals distinctive distributions of spectrin and dystrophin. Eur J Cell Biol. 2005; 84(12):961-71. DOI: 10.1016/j.ejcb.2005.09.015. View

19.

Rash J, Olson C, Pouliot W, Davidson K, Yasumura T, Furman C . Connexin36 vs. connexin32, "miniature" neuronal gap junctions, and limited electrotonic coupling in rodent suprachiasmatic nucleus. Neuroscience. 2007; 149(2):350-71. PMC: 2245895. DOI: 10.1016/j.neuroscience.2007.06.052. View

20.

Robenek H, Robenek M, Buers I, Lorkowski S, Hofnagel O, Troyer D . Lipid droplets gain PAT family proteins by interaction with specialized plasma membrane domains. J Biol Chem. 2005; 280(28):26330-8. DOI: 10.1074/jbc.M413312200. View