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Recent advances in freeze-fracture electron microscopy: the replica immunolabeling technique.

Robenek H, Severs NJ - Biol Proced Online (2008)

Bottom Line: Freeze-fracture electron microscopy is a technique for examining the ultrastructure of rapidly frozen biological samples by transmission electron microscopy.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.

View Article: PubMed Central - PubMed

Affiliation: University of Münster, Domagkstr. 3D-48149 Münster, Germany.

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.

No MeSH data available.


Related in: MedlinePlus

Fig. 5                  Similar view to Fig 4. at higher magnification. Gold label for adipophilin is clearly seen on the ER membrane (P Face, PF) immediately adjacent to the lipid droplets (arrows). The lower example arrowed shows a clear demonstration of how the ER membrane curves around the contour of the lipid droplet.
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f5: Fig. 5 Similar view to Fig 4. at higher magnification. Gold label for adipophilin is clearly seen on the ER membrane (P Face, PF) immediately adjacent to the lipid droplets (arrows). The lower example arrowed shows a clear demonstration of how the ER membrane curves around the contour of the lipid droplet.

Mentions: Results from FRIL refute this model on several counts (21). First, freeze fracture, by permitting unique three-dimensional views of the spatial relationships of membranes and organelles, demonstrates that at sites of close association, the lipid droplet is not situated within the ER membrane, but adjacent to it (Figs. 4-6). Both ER membranes clearly lie external to and follow the contour of the lipid droplet (Figs. 4, 5), enclosing it in a manner akin to an egg-cup (the ER) holding an egg (the lipid droplet) (Fig. 6A-C). Application of FRIL demonstrates that the PAT family protein, adipophilin, is concentrated in prominent clusters in the P half of the ER membrane at the site of the closely apposed lipid droplet (Fig. 6A, B), as well as in the lipid droplet surface apposed to the ER (Fig. 6D, E). Adipophilin is thus positioned to play a role in lipid droplet growth by facilitating lipid transfer from the ER to the droplet. FRIL demonstrates unequivocally that lipid droplets develop adjacent and external to specialized domains of the ER membrane enriched in adipophilin, not within the bilayer of the ER as previously supposed.


Recent advances in freeze-fracture electron microscopy: the replica immunolabeling technique.

Robenek H, Severs NJ - Biol Proced Online (2008)

Fig. 5                  Similar view to Fig 4. at higher magnification. Gold label for adipophilin is clearly seen on the ER membrane (P Face, PF) immediately adjacent to the lipid droplets (arrows). The lower example arrowed shows a clear demonstration of how the ER membrane curves around the contour of the lipid droplet.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2275045&req=5

f5: Fig. 5 Similar view to Fig 4. at higher magnification. Gold label for adipophilin is clearly seen on the ER membrane (P Face, PF) immediately adjacent to the lipid droplets (arrows). The lower example arrowed shows a clear demonstration of how the ER membrane curves around the contour of the lipid droplet.
Mentions: Results from FRIL refute this model on several counts (21). First, freeze fracture, by permitting unique three-dimensional views of the spatial relationships of membranes and organelles, demonstrates that at sites of close association, the lipid droplet is not situated within the ER membrane, but adjacent to it (Figs. 4-6). Both ER membranes clearly lie external to and follow the contour of the lipid droplet (Figs. 4, 5), enclosing it in a manner akin to an egg-cup (the ER) holding an egg (the lipid droplet) (Fig. 6A-C). Application of FRIL demonstrates that the PAT family protein, adipophilin, is concentrated in prominent clusters in the P half of the ER membrane at the site of the closely apposed lipid droplet (Fig. 6A, B), as well as in the lipid droplet surface apposed to the ER (Fig. 6D, E). Adipophilin is thus positioned to play a role in lipid droplet growth by facilitating lipid transfer from the ER to the droplet. FRIL demonstrates unequivocally that lipid droplets develop adjacent and external to specialized domains of the ER membrane enriched in adipophilin, not within the bilayer of the ER as previously supposed.

Bottom Line: Freeze-fracture electron microscopy is a technique for examining the ultrastructure of rapidly frozen biological samples by transmission electron microscopy.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.

View Article: PubMed Central - PubMed

Affiliation: University of Münster, Domagkstr. 3D-48149 Münster, Germany.

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.

No MeSH data available.


Related in: MedlinePlus