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The use of time-resolved fluorescence imaging in the study of protein kinase C localisation in cells.

Stubbs CD, Botchway SW, Slater SJ, Parker AW - BMC Cell Biol. (2005)

Bottom Line: PKCalpha is found widely in the cytoplasm and nucleus in most cells.Based on the extent of lifetime quenching observed, the results are consistent with a direct interaction between PKCalpha and caveolin in the endosomes, and possibly an indirect interaction in the peripheral regions of the cell.The results show that 2P-FLIM-FRET imaging offers an approach that can provide information not only confirming the occurrence of specific protein-protein interactions but where they occur within the cell.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA. cstubbs@stubbsmail.com

ABSTRACT

Background: Two-photon-excitation fluorescence lifetime imaging (2P-FLIM) was used to investigate the association of protein kinase C alpha (PKCalpha) with caveolin in CHO cells. PKCalpha is found widely in the cytoplasm and nucleus in most cells. Upon activation, as a result of increased intracellular Ca2+ and production of DAG, through G-protein coupled-phospholipase C signalling, PKC translocates to a variety of regions in the cell where it phosphorylates and interacts with many signalling pathways. Due to its wide distribution, discerning a particular interaction from others within the cell is extremely difficult.

Results: Fluorescence energy transfer (FRET), between GFP-PKCalpha and DsRed-caveolin, was used to investigate the interaction between caveolin and PKC, an aspect of signalling that is poorly understood. Using 2P-FLIM measurements, the lifetime of GFP was found to decrease (quench) in certain regions of the cell from approximately 2.2 ns to approximately 1.5 ns when the GFP and DsRed were sufficiently close for FRET to occur. This only occurred when intracellular Ca2+ increased or in the presence of phorbol ester, and was an indication of PKC and caveolin co-localisation under these conditions. In the case of phorbol ester stimulated PKC translocation, as commonly used to model PKC activation, three PKC areas could be delineated. These included PKCalpha that was not associated with caveolin in the nucleus and cytoplasm, PKCalpha associated with caveolin in the cytoplasm/perinuclear regions and probably in endosomes, and PKC in the peripheral regions of the cell, possibly indirectly interacting with caveolin.

Conclusion: Based on the extent of lifetime quenching observed, the results are consistent with a direct interaction between PKCalpha and caveolin in the endosomes, and possibly an indirect interaction in the peripheral regions of the cell. The results show that 2P-FLIM-FRET imaging offers an approach that can provide information not only confirming the occurrence of specific protein-protein interactions but where they occur within the cell.

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Lifetime imaging of GFP-PKC co-expressed with DsRed-cav in CHO cells: effect of phorbol ester. Epifluorescence and 2P-FLIM images were collected as described in the legend to Figure 2. Cells were treated with TPA (100 nM) for 3 min before mounting and fixation as described in Methods. (a) left: GFP-PKC and DsRed-cav co-distribution revealed in a green and red epifluorescence image (red and green filters) and right: DsRed-cav visualised using a red filter, the caveolin was mainly restricted to the perinuclear region with PKC more widely distributed. Three distinct GFP lifetimes are discernable in the lifetime image and were separately analysed. For the lifetime images shown in (b, c and d), representative single point analyses within the regions enclosed by dashed white lines are analysed in (f, g and h) respectively, as follows: (b and f) peripheral regions (τ avg: 1.8 ns; single point 1.87 ns [χ2 1.00]); (c and g) the nuclear region (τ avg: 2.0 ns; single point 2.00 [χ2 1.00]); (d and h) the cytoplasm (τ avg: 1.5 ns; single point 1.48 ns [χ2 1.56]). (e) example of derivation of average lifetime for one of the three areas, shown for the cytoplasm, with the analysis for the area enclosed in red (lifetime colour coding shown in the inset) and other similar areas in the cytoplasm indicated by white arrows. Cells shown are representative images from replicate experiments.
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Figure 6: Lifetime imaging of GFP-PKC co-expressed with DsRed-cav in CHO cells: effect of phorbol ester. Epifluorescence and 2P-FLIM images were collected as described in the legend to Figure 2. Cells were treated with TPA (100 nM) for 3 min before mounting and fixation as described in Methods. (a) left: GFP-PKC and DsRed-cav co-distribution revealed in a green and red epifluorescence image (red and green filters) and right: DsRed-cav visualised using a red filter, the caveolin was mainly restricted to the perinuclear region with PKC more widely distributed. Three distinct GFP lifetimes are discernable in the lifetime image and were separately analysed. For the lifetime images shown in (b, c and d), representative single point analyses within the regions enclosed by dashed white lines are analysed in (f, g and h) respectively, as follows: (b and f) peripheral regions (τ avg: 1.8 ns; single point 1.87 ns [χ2 1.00]); (c and g) the nuclear region (τ avg: 2.0 ns; single point 2.00 [χ2 1.00]); (d and h) the cytoplasm (τ avg: 1.5 ns; single point 1.48 ns [χ2 1.56]). (e) example of derivation of average lifetime for one of the three areas, shown for the cytoplasm, with the analysis for the area enclosed in red (lifetime colour coding shown in the inset) and other similar areas in the cytoplasm indicated by white arrows. Cells shown are representative images from replicate experiments.

Mentions: The treatment of the cells with the phorbol ester TPA not only induces translocation of PKC to different cell compartments but also produces catalytically active PKC. In general, PKC moves to the outer membrane and to perinuclear regions, and associates with various signalling and cytostructural components in the cell. The lifetime image data revealed that, similar to the effect of increasing intracellular Ca2+, phorbol ester induced PKC to associate with caveolin, the GFP lifetime being reduced accordingly, as shown in Figure 6.


The use of time-resolved fluorescence imaging in the study of protein kinase C localisation in cells.

Stubbs CD, Botchway SW, Slater SJ, Parker AW - BMC Cell Biol. (2005)

Lifetime imaging of GFP-PKC co-expressed with DsRed-cav in CHO cells: effect of phorbol ester. Epifluorescence and 2P-FLIM images were collected as described in the legend to Figure 2. Cells were treated with TPA (100 nM) for 3 min before mounting and fixation as described in Methods. (a) left: GFP-PKC and DsRed-cav co-distribution revealed in a green and red epifluorescence image (red and green filters) and right: DsRed-cav visualised using a red filter, the caveolin was mainly restricted to the perinuclear region with PKC more widely distributed. Three distinct GFP lifetimes are discernable in the lifetime image and were separately analysed. For the lifetime images shown in (b, c and d), representative single point analyses within the regions enclosed by dashed white lines are analysed in (f, g and h) respectively, as follows: (b and f) peripheral regions (τ avg: 1.8 ns; single point 1.87 ns [χ2 1.00]); (c and g) the nuclear region (τ avg: 2.0 ns; single point 2.00 [χ2 1.00]); (d and h) the cytoplasm (τ avg: 1.5 ns; single point 1.48 ns [χ2 1.56]). (e) example of derivation of average lifetime for one of the three areas, shown for the cytoplasm, with the analysis for the area enclosed in red (lifetime colour coding shown in the inset) and other similar areas in the cytoplasm indicated by white arrows. Cells shown are representative images from replicate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Lifetime imaging of GFP-PKC co-expressed with DsRed-cav in CHO cells: effect of phorbol ester. Epifluorescence and 2P-FLIM images were collected as described in the legend to Figure 2. Cells were treated with TPA (100 nM) for 3 min before mounting and fixation as described in Methods. (a) left: GFP-PKC and DsRed-cav co-distribution revealed in a green and red epifluorescence image (red and green filters) and right: DsRed-cav visualised using a red filter, the caveolin was mainly restricted to the perinuclear region with PKC more widely distributed. Three distinct GFP lifetimes are discernable in the lifetime image and were separately analysed. For the lifetime images shown in (b, c and d), representative single point analyses within the regions enclosed by dashed white lines are analysed in (f, g and h) respectively, as follows: (b and f) peripheral regions (τ avg: 1.8 ns; single point 1.87 ns [χ2 1.00]); (c and g) the nuclear region (τ avg: 2.0 ns; single point 2.00 [χ2 1.00]); (d and h) the cytoplasm (τ avg: 1.5 ns; single point 1.48 ns [χ2 1.56]). (e) example of derivation of average lifetime for one of the three areas, shown for the cytoplasm, with the analysis for the area enclosed in red (lifetime colour coding shown in the inset) and other similar areas in the cytoplasm indicated by white arrows. Cells shown are representative images from replicate experiments.
Mentions: The treatment of the cells with the phorbol ester TPA not only induces translocation of PKC to different cell compartments but also produces catalytically active PKC. In general, PKC moves to the outer membrane and to perinuclear regions, and associates with various signalling and cytostructural components in the cell. The lifetime image data revealed that, similar to the effect of increasing intracellular Ca2+, phorbol ester induced PKC to associate with caveolin, the GFP lifetime being reduced accordingly, as shown in Figure 6.

Bottom Line: PKCalpha is found widely in the cytoplasm and nucleus in most cells.Based on the extent of lifetime quenching observed, the results are consistent with a direct interaction between PKCalpha and caveolin in the endosomes, and possibly an indirect interaction in the peripheral regions of the cell.The results show that 2P-FLIM-FRET imaging offers an approach that can provide information not only confirming the occurrence of specific protein-protein interactions but where they occur within the cell.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA. cstubbs@stubbsmail.com

ABSTRACT

Background: Two-photon-excitation fluorescence lifetime imaging (2P-FLIM) was used to investigate the association of protein kinase C alpha (PKCalpha) with caveolin in CHO cells. PKCalpha is found widely in the cytoplasm and nucleus in most cells. Upon activation, as a result of increased intracellular Ca2+ and production of DAG, through G-protein coupled-phospholipase C signalling, PKC translocates to a variety of regions in the cell where it phosphorylates and interacts with many signalling pathways. Due to its wide distribution, discerning a particular interaction from others within the cell is extremely difficult.

Results: Fluorescence energy transfer (FRET), between GFP-PKCalpha and DsRed-caveolin, was used to investigate the interaction between caveolin and PKC, an aspect of signalling that is poorly understood. Using 2P-FLIM measurements, the lifetime of GFP was found to decrease (quench) in certain regions of the cell from approximately 2.2 ns to approximately 1.5 ns when the GFP and DsRed were sufficiently close for FRET to occur. This only occurred when intracellular Ca2+ increased or in the presence of phorbol ester, and was an indication of PKC and caveolin co-localisation under these conditions. In the case of phorbol ester stimulated PKC translocation, as commonly used to model PKC activation, three PKC areas could be delineated. These included PKCalpha that was not associated with caveolin in the nucleus and cytoplasm, PKCalpha associated with caveolin in the cytoplasm/perinuclear regions and probably in endosomes, and PKC in the peripheral regions of the cell, possibly indirectly interacting with caveolin.

Conclusion: Based on the extent of lifetime quenching observed, the results are consistent with a direct interaction between PKCalpha and caveolin in the endosomes, and possibly an indirect interaction in the peripheral regions of the cell. The results show that 2P-FLIM-FRET imaging offers an approach that can provide information not only confirming the occurrence of specific protein-protein interactions but where they occur within the cell.

Show MeSH