Limits...
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

Photoconversion of GFP from green to red state in vivo.Single snapshots from the pre-photoconversion and post-photoconversion are shown. (a) Tobacco suspension cell culture in which GFP is targeted to cytosol. Photoconverted GFP spreads through the cytosol via cytoplasmic strands. Photoconversion was performed at 50% laser power and 30 iterations of 111 milliseconds (ms). (b) Schematic representation of a tobacco suspension culture cell. The cell wall (brown) surrounds the plasma membrane (red). A large vacuole (blue) occupies the majority of the space inside the cell. The endoplasmic reticulum network (gray) surrounds the vacuole and the nucleus (white) and extends through the rest of the cytosol (white). Cytoplasmic strands exist as extensions of the cytosol between organelles. (c) Changes in fluorescence intensity in the irradiated area in (a) over time indicate increasing red and decreasing green fluorescence. (d) Drosophila gut cells from 3rd instar larvae expressing S65T-GFP. (e) Rat PC12 cells expressing EGFP fusion to exon 1 of human HttQ103 gene. White arrowheads highlight cytosolic inclusions. Images acquired by 30 iterations with the duration of 190 ms each. Laser power was adjusted at 70%. Bars 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4493561&req=5

f4: Photoconversion of GFP from green to red state in vivo.Single snapshots from the pre-photoconversion and post-photoconversion are shown. (a) Tobacco suspension cell culture in which GFP is targeted to cytosol. Photoconverted GFP spreads through the cytosol via cytoplasmic strands. Photoconversion was performed at 50% laser power and 30 iterations of 111 milliseconds (ms). (b) Schematic representation of a tobacco suspension culture cell. The cell wall (brown) surrounds the plasma membrane (red). A large vacuole (blue) occupies the majority of the space inside the cell. The endoplasmic reticulum network (gray) surrounds the vacuole and the nucleus (white) and extends through the rest of the cytosol (white). Cytoplasmic strands exist as extensions of the cytosol between organelles. (c) Changes in fluorescence intensity in the irradiated area in (a) over time indicate increasing red and decreasing green fluorescence. (d) Drosophila gut cells from 3rd instar larvae expressing S65T-GFP. (e) Rat PC12 cells expressing EGFP fusion to exon 1 of human HttQ103 gene. White arrowheads highlight cytosolic inclusions. Images acquired by 30 iterations with the duration of 190 ms each. Laser power was adjusted at 70%. Bars 10 μm.

Mentions: To determine whether GFP could be converted from the green to the red state in vivo, we performed photoconversion experiments in transgenic tobacco cells in which GFP was localized in the cytosol19 (Fig. 4a-c). Converting GFP from green to red state resulted in rapid diffusion of the red form within the plant cytosol. In less than 3 seconds after irradiation, red-GFP could be detected in the red channel. The photoconverted red-GFP then diffused through the whole cell and was readily visible throughout the cell within 2 minutes (Fig. 4a and Supplementary Movie S2). Fluorescence intensity was measured in the area indicated by a white circle in the irradiated area. There is a noticeable decrease in intensity of the green signal and a concomitant increase in intensity of the red signal, which is an indication of photoconversion from green to red state (Fig. 4c). We detected no red signal when plant cells not expressing GFP were irradiated and imaged under the same conditions.


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

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

Photoconversion of GFP from green to red state in vivo.Single snapshots from the pre-photoconversion and post-photoconversion are shown. (a) Tobacco suspension cell culture in which GFP is targeted to cytosol. Photoconverted GFP spreads through the cytosol via cytoplasmic strands. Photoconversion was performed at 50% laser power and 30 iterations of 111 milliseconds (ms). (b) Schematic representation of a tobacco suspension culture cell. The cell wall (brown) surrounds the plasma membrane (red). A large vacuole (blue) occupies the majority of the space inside the cell. The endoplasmic reticulum network (gray) surrounds the vacuole and the nucleus (white) and extends through the rest of the cytosol (white). Cytoplasmic strands exist as extensions of the cytosol between organelles. (c) Changes in fluorescence intensity in the irradiated area in (a) over time indicate increasing red and decreasing green fluorescence. (d) Drosophila gut cells from 3rd instar larvae expressing S65T-GFP. (e) Rat PC12 cells expressing EGFP fusion to exon 1 of human HttQ103 gene. White arrowheads highlight cytosolic inclusions. Images acquired by 30 iterations with the duration of 190 ms each. Laser power was adjusted at 70%. Bars 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Photoconversion of GFP from green to red state in vivo.Single snapshots from the pre-photoconversion and post-photoconversion are shown. (a) Tobacco suspension cell culture in which GFP is targeted to cytosol. Photoconverted GFP spreads through the cytosol via cytoplasmic strands. Photoconversion was performed at 50% laser power and 30 iterations of 111 milliseconds (ms). (b) Schematic representation of a tobacco suspension culture cell. The cell wall (brown) surrounds the plasma membrane (red). A large vacuole (blue) occupies the majority of the space inside the cell. The endoplasmic reticulum network (gray) surrounds the vacuole and the nucleus (white) and extends through the rest of the cytosol (white). Cytoplasmic strands exist as extensions of the cytosol between organelles. (c) Changes in fluorescence intensity in the irradiated area in (a) over time indicate increasing red and decreasing green fluorescence. (d) Drosophila gut cells from 3rd instar larvae expressing S65T-GFP. (e) Rat PC12 cells expressing EGFP fusion to exon 1 of human HttQ103 gene. White arrowheads highlight cytosolic inclusions. Images acquired by 30 iterations with the duration of 190 ms each. Laser power was adjusted at 70%. Bars 10 μm.
Mentions: To determine whether GFP could be converted from the green to the red state in vivo, we performed photoconversion experiments in transgenic tobacco cells in which GFP was localized in the cytosol19 (Fig. 4a-c). Converting GFP from green to red state resulted in rapid diffusion of the red form within the plant cytosol. In less than 3 seconds after irradiation, red-GFP could be detected in the red channel. The photoconverted red-GFP then diffused through the whole cell and was readily visible throughout the cell within 2 minutes (Fig. 4a and Supplementary Movie S2). Fluorescence intensity was measured in the area indicated by a white circle in the irradiated area. There is a noticeable decrease in intensity of the green signal and a concomitant increase in intensity of the red signal, which is an indication of photoconversion from green to red state (Fig. 4c). We detected no red signal when plant cells not expressing GFP were irradiated and imaged under the same conditions.

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