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Green to red photoconversion of GFP for protein tracking in vivo.

Sattarzadeh A, Saberianfar R, Zipfel WR, Menassa R, Hanson MR - Sci Rep (2015)

Bottom Line: A variety of fluorescent proteins have been identified that undergo shifts in spectral emission properties over time or once they are irradiated by ultraviolet or blue light.However, before genes encoding these fluorescent proteins were available, many proteins have already been labelled with GFP in transgenic cells; a number of model organisms feature collections of GFP-tagged lines and organisms.We demonstrate its use in transgenic plant, Drosophila and mammalian cells in vivo.

View Article: PubMed Central - PubMed

Affiliation: Cornell University, Department of Molecular Biology and Genetics, Ithaca, NY, 14853 USA.

ABSTRACT
A variety of fluorescent proteins have been identified that undergo shifts in spectral emission properties over time or once they are irradiated by ultraviolet or blue light. Such proteins are finding application in following the dynamics of particular proteins or labelled organelles within the cell. However, before genes encoding these fluorescent proteins were available, many proteins have already been labelled with GFP in transgenic cells; a number of model organisms feature collections of GFP-tagged lines and organisms. Here we describe a fast, localized and non-invasive method for GFP photoconversion from green to red. We demonstrate its use in transgenic plant, Drosophila and mammalian cells in vivo. While genes encoding fluorescent proteins specifically designed for photoconversion will usually be advantageous when creating new transgenic lines, our method for photoconversion of GFP allows the use of existing GFP-tagged transgenic lines for studies of dynamic processes in living cells.

No MeSH data available.


Related in: MedlinePlus

EGFP photoconversion occurs in N. benthamiana cells transiently expressing EGFP.(a) ER-targeted EGFP photoconverts from green to red upon excitation with 405-nm laser at 60% of laser power and 100 iterations with duration of 145 milliseconds each. The photoconverted red protein travels within the ER network of the cell. (b) Cytosolic EGFP photoconverts and spreads within the cytosolic space of the cell. Photoconversion was performed at 70% of laser power and 30 iterations with duration of 190 milliseconds each. White circles represent the irradiated area. Bar 20 μm. (c) Schematic representation of a Nicotiana benthamiana epidermal leaf cell. Puzzle-shaped epidermal cells are surrounded with the cell wall (brown). The space between the cell walls of two neighboring cells, apoplast, is shown in green. The plasma membrane (red) surrounds the cell. A large vacuole (blue) occupies the majority of the inner space of the cell. Endoplasmic reticulum (black) is localized around the nucleus and spreads throughout the cytosol (white), which is pushed against the plasma membrane by the vacuole. Chloroplasts are drawn in black as oval structures in the cytosol.
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f5: EGFP photoconversion occurs in N. benthamiana cells transiently expressing EGFP.(a) ER-targeted EGFP photoconverts from green to red upon excitation with 405-nm laser at 60% of laser power and 100 iterations with duration of 145 milliseconds each. The photoconverted red protein travels within the ER network of the cell. (b) Cytosolic EGFP photoconverts and spreads within the cytosolic space of the cell. Photoconversion was performed at 70% of laser power and 30 iterations with duration of 190 milliseconds each. White circles represent the irradiated area. Bar 20 μm. (c) Schematic representation of a Nicotiana benthamiana epidermal leaf cell. Puzzle-shaped epidermal cells are surrounded with the cell wall (brown). The space between the cell walls of two neighboring cells, apoplast, is shown in green. The plasma membrane (red) surrounds the cell. A large vacuole (blue) occupies the majority of the inner space of the cell. Endoplasmic reticulum (black) is localized around the nucleus and spreads throughout the cytosol (white), which is pushed against the plasma membrane by the vacuole. Chloroplasts are drawn in black as oval structures in the cytosol.

Mentions: Transient expression methods are also frequently used to study the subcellular localization of GFP-tagged proteins. In plants, several methods exist for transient expression, including particle bombardment, infiltration of Agrobacterium tumefaciens (Agroinfiltration), or protoplast transfection. In addition to photoconversion of stably transformed tobacco cells, we were able to photoconvert EGFP that was transiently expressed in Nicotiana benthamiana epidermal leaf cells by Agroinfiltration (Fig. 5, Supplementary Movies S3-4). We were able to label the endoplasmic reticulum (ER) with GFP targeted to the ER and observe movement through the ER (Fig. 5a). Also, GFP located in the cytosol could be photoconverted and we could follow its movement through the cell (Fig. 5b).


Green to red photoconversion of GFP for protein tracking in vivo.

Sattarzadeh A, Saberianfar R, Zipfel WR, Menassa R, Hanson MR - Sci Rep (2015)

EGFP photoconversion occurs in N. benthamiana cells transiently expressing EGFP.(a) ER-targeted EGFP photoconverts from green to red upon excitation with 405-nm laser at 60% of laser power and 100 iterations with duration of 145 milliseconds each. The photoconverted red protein travels within the ER network of the cell. (b) Cytosolic EGFP photoconverts and spreads within the cytosolic space of the cell. Photoconversion was performed at 70% of laser power and 30 iterations with duration of 190 milliseconds each. White circles represent the irradiated area. Bar 20 μm. (c) Schematic representation of a Nicotiana benthamiana epidermal leaf cell. Puzzle-shaped epidermal cells are surrounded with the cell wall (brown). The space between the cell walls of two neighboring cells, apoplast, is shown in green. The plasma membrane (red) surrounds the cell. A large vacuole (blue) occupies the majority of the inner space of the cell. Endoplasmic reticulum (black) is localized around the nucleus and spreads throughout the cytosol (white), which is pushed against the plasma membrane by the vacuole. Chloroplasts are drawn in black as oval structures in the cytosol.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493561&req=5

f5: EGFP photoconversion occurs in N. benthamiana cells transiently expressing EGFP.(a) ER-targeted EGFP photoconverts from green to red upon excitation with 405-nm laser at 60% of laser power and 100 iterations with duration of 145 milliseconds each. The photoconverted red protein travels within the ER network of the cell. (b) Cytosolic EGFP photoconverts and spreads within the cytosolic space of the cell. Photoconversion was performed at 70% of laser power and 30 iterations with duration of 190 milliseconds each. White circles represent the irradiated area. Bar 20 μm. (c) Schematic representation of a Nicotiana benthamiana epidermal leaf cell. Puzzle-shaped epidermal cells are surrounded with the cell wall (brown). The space between the cell walls of two neighboring cells, apoplast, is shown in green. The plasma membrane (red) surrounds the cell. A large vacuole (blue) occupies the majority of the inner space of the cell. Endoplasmic reticulum (black) is localized around the nucleus and spreads throughout the cytosol (white), which is pushed against the plasma membrane by the vacuole. Chloroplasts are drawn in black as oval structures in the cytosol.
Mentions: Transient expression methods are also frequently used to study the subcellular localization of GFP-tagged proteins. In plants, several methods exist for transient expression, including particle bombardment, infiltration of Agrobacterium tumefaciens (Agroinfiltration), or protoplast transfection. In addition to photoconversion of stably transformed tobacco cells, we were able to photoconvert EGFP that was transiently expressed in Nicotiana benthamiana epidermal leaf cells by Agroinfiltration (Fig. 5, Supplementary Movies S3-4). We were able to label the endoplasmic reticulum (ER) with GFP targeted to the ER and observe movement through the ER (Fig. 5a). Also, GFP located in the cytosol could be photoconverted and we could follow its movement through the cell (Fig. 5b).

Bottom Line: A variety of fluorescent proteins have been identified that undergo shifts in spectral emission properties over time or once they are irradiated by ultraviolet or blue light.However, before genes encoding these fluorescent proteins were available, many proteins have already been labelled with GFP in transgenic cells; a number of model organisms feature collections of GFP-tagged lines and organisms.We demonstrate its use in transgenic plant, Drosophila and mammalian cells in vivo.

View Article: PubMed Central - PubMed

Affiliation: Cornell University, Department of Molecular Biology and Genetics, Ithaca, NY, 14853 USA.

ABSTRACT
A variety of fluorescent proteins have been identified that undergo shifts in spectral emission properties over time or once they are irradiated by ultraviolet or blue light. Such proteins are finding application in following the dynamics of particular proteins or labelled organelles within the cell. However, before genes encoding these fluorescent proteins were available, many proteins have already been labelled with GFP in transgenic cells; a number of model organisms feature collections of GFP-tagged lines and organisms. Here we describe a fast, localized and non-invasive method for GFP photoconversion from green to red. We demonstrate its use in transgenic plant, Drosophila and mammalian cells in vivo. While genes encoding fluorescent proteins specifically designed for photoconversion will usually be advantageous when creating new transgenic lines, our method for photoconversion of GFP allows the use of existing GFP-tagged transgenic lines for studies of dynamic processes in living cells.

No MeSH data available.


Related in: MedlinePlus