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GFP-tagged proteins visualized by freeze-fracture immuno-electron microscopy: a new tool in cellular and molecular medicine.

Robenek H, Buers I, Hofnagel O, Lorkowski S, Severs NJ - J. Cell. Mol. Med. (2008)

Bottom Line: Here we demonstrate that the electron microscopic technique freeze-fracture replica immunogold labelling overcomes these disadvantages and can be used to define, at high resolution, the precise location of GFP-tagged proteins in specific membrane systems and organelles of the cell.Moreover, this technique provides information on the location of the protein within the phospholipid bilayer, potentially providing insight into mis-orientation of tagged proteins compared to their untagged counterparts.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.

View Article: PubMed Central - PubMed

Affiliation: Leibniz Institute for Arteriosclerosis Research, University of Münster, Münster, Germany. robenek@uni-muenster.de

ABSTRACT
GFP-tagging is widely used as a molecular tool to localize and visualize the trafficking of proteins in cells but interpretation is frequently limited by the low resolution afforded by fluorescence light microscopy. Although complementary thin-section immunogold electron microscopic techniques go some way in aiding interpretation, major limitations, such as relatively poor structural preservation of membrane systems, low labelling efficiency and the two-dimensional nature of the images, remain. Here we demonstrate that the electron microscopic technique freeze-fracture replica immunogold labelling overcomes these disadvantages and can be used to define, at high resolution, the precise location of GFP-tagged proteins in specific membrane systems and organelles of the cell. Moreover, this technique provides information on the location of the protein within the phospholipid bilayer, potentially providing insight into mis-orientation of tagged proteins compared to their untagged counterparts. Complementary application of the freeze-fracture replica immunogold labelling technique alongside conventional fluorescence microscopy is seen as a novel and valuable approach to verification, clarification and extension of the data obtained using fluorescent-tagged proteins. 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.

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Related in: MedlinePlus

The GFP-adipophilin (ADFP) is also detected in the nuclear membranes by labelling with adipophilin antibodies (A), GFP antibodies (B) and both antibodies (18 nm gold particles encircled) (C). NP, nuclear pores; oM, outer nuclear membrane; iM, inner nuclear membrane; PF, fracture face of half-membrane leaflet attached to protoplasm; EF, fracture face of half-membrane leaflet attached to endo-plasmic space (lumen between inner and outer nuclear membranes or endoplasmic reticulum). Bars: 0.2 μm
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fig07: The GFP-adipophilin (ADFP) is also detected in the nuclear membranes by labelling with adipophilin antibodies (A), GFP antibodies (B) and both antibodies (18 nm gold particles encircled) (C). NP, nuclear pores; oM, outer nuclear membrane; iM, inner nuclear membrane; PF, fracture face of half-membrane leaflet attached to protoplasm; EF, fracture face of half-membrane leaflet attached to endo-plasmic space (lumen between inner and outer nuclear membranes or endoplasmic reticulum). Bars: 0.2 μm

Mentions: In NPC cells transfected with adipophilin-GFP, the pattern of labelling on lipid droplets is similar to that observed with the GFP-perilipin transfected cells, irrespective of whether anti-GFP or anti-adipophilin antibodies are applied (Fig. 5). However, in addition, prominent labelling for adipophilin-GFP is apparent in the plasma membrane and nuclear membranes (Figs. 6 and 7). In the plasma membrane, a high density of labelling is present on the P-half of the membrane (the half membrane leaflet adjacent to the protoplasm) though significant labelling is also apparent on the E-half (the half membrane leaflet adjacent to the extracellular space) (Fig. 6). This finding suggests that the presence of the GFP-tag results in an abnormal orientation of adipophilin in the plasma membrane, for normally this protein only occupies the P-half [18, 20]. A similar observation was made in the nuclear membranes, suggesting that GFP induces mis-orientation of the adipophilin irrespective of the membrane destination. Thus, the freeze-fracture immunolabelling approach, in contrast to other high-resolution localization techniques, has the ability to identify alterations in the orientation of tagged membrane proteins.


GFP-tagged proteins visualized by freeze-fracture immuno-electron microscopy: a new tool in cellular and molecular medicine.

Robenek H, Buers I, Hofnagel O, Lorkowski S, Severs NJ - J. Cell. Mol. Med. (2008)

The GFP-adipophilin (ADFP) is also detected in the nuclear membranes by labelling with adipophilin antibodies (A), GFP antibodies (B) and both antibodies (18 nm gold particles encircled) (C). NP, nuclear pores; oM, outer nuclear membrane; iM, inner nuclear membrane; PF, fracture face of half-membrane leaflet attached to protoplasm; EF, fracture face of half-membrane leaflet attached to endo-plasmic space (lumen between inner and outer nuclear membranes or endoplasmic reticulum). Bars: 0.2 μm
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4496151&req=5

fig07: The GFP-adipophilin (ADFP) is also detected in the nuclear membranes by labelling with adipophilin antibodies (A), GFP antibodies (B) and both antibodies (18 nm gold particles encircled) (C). NP, nuclear pores; oM, outer nuclear membrane; iM, inner nuclear membrane; PF, fracture face of half-membrane leaflet attached to protoplasm; EF, fracture face of half-membrane leaflet attached to endo-plasmic space (lumen between inner and outer nuclear membranes or endoplasmic reticulum). Bars: 0.2 μm
Mentions: In NPC cells transfected with adipophilin-GFP, the pattern of labelling on lipid droplets is similar to that observed with the GFP-perilipin transfected cells, irrespective of whether anti-GFP or anti-adipophilin antibodies are applied (Fig. 5). However, in addition, prominent labelling for adipophilin-GFP is apparent in the plasma membrane and nuclear membranes (Figs. 6 and 7). In the plasma membrane, a high density of labelling is present on the P-half of the membrane (the half membrane leaflet adjacent to the protoplasm) though significant labelling is also apparent on the E-half (the half membrane leaflet adjacent to the extracellular space) (Fig. 6). This finding suggests that the presence of the GFP-tag results in an abnormal orientation of adipophilin in the plasma membrane, for normally this protein only occupies the P-half [18, 20]. A similar observation was made in the nuclear membranes, suggesting that GFP induces mis-orientation of the adipophilin irrespective of the membrane destination. Thus, the freeze-fracture immunolabelling approach, in contrast to other high-resolution localization techniques, has the ability to identify alterations in the orientation of tagged membrane proteins.

Bottom Line: Here we demonstrate that the electron microscopic technique freeze-fracture replica immunogold labelling overcomes these disadvantages and can be used to define, at high resolution, the precise location of GFP-tagged proteins in specific membrane systems and organelles of the cell.Moreover, this technique provides information on the location of the protein within the phospholipid bilayer, potentially providing insight into mis-orientation of tagged proteins compared to their untagged counterparts.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.

View Article: PubMed Central - PubMed

Affiliation: Leibniz Institute for Arteriosclerosis Research, University of Münster, Münster, Germany. robenek@uni-muenster.de

ABSTRACT
GFP-tagging is widely used as a molecular tool to localize and visualize the trafficking of proteins in cells but interpretation is frequently limited by the low resolution afforded by fluorescence light microscopy. Although complementary thin-section immunogold electron microscopic techniques go some way in aiding interpretation, major limitations, such as relatively poor structural preservation of membrane systems, low labelling efficiency and the two-dimensional nature of the images, remain. Here we demonstrate that the electron microscopic technique freeze-fracture replica immunogold labelling overcomes these disadvantages and can be used to define, at high resolution, the precise location of GFP-tagged proteins in specific membrane systems and organelles of the cell. Moreover, this technique provides information on the location of the protein within the phospholipid bilayer, potentially providing insight into mis-orientation of tagged proteins compared to their untagged counterparts. Complementary application of the freeze-fracture replica immunogold labelling technique alongside conventional fluorescence microscopy is seen as a novel and valuable approach to verification, clarification and extension of the data obtained using fluorescent-tagged proteins. 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.

Show MeSH
Related in: MedlinePlus