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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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Fluorescence lifetime imaging of GFP-PKC expressed in CHO cells. GFP-PKC was transiently expressed in CHO cells as described in the legend in Figure 1 and detailed under Methods. Images were acquired using a Nikon 2000 inverted epifluorescence microscope with a 60× objective and GFP/DsRed filter sets and an ICAM camera. The 2P-FLIM images were collected using the TCSPC fast scanning imaging mode as described under Methods. (a) Conventional epifluorescence image of GFP-PKC distribution in a resting CHO cell, (b) lifetime image of the same cell with the analysis area enclosed by the red line (cytosol) shown (with colour coding) in the inset and giving an average lifetime of ~2.2 ns and (c) with the analysis area enclosed by the red line (nucleus) shown in the inset giving an average lifetime of ~2.0 ns. Cells shown are representative images from replicate experiments.
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Figure 2: Fluorescence lifetime imaging of GFP-PKC expressed in CHO cells. GFP-PKC was transiently expressed in CHO cells as described in the legend in Figure 1 and detailed under Methods. Images were acquired using a Nikon 2000 inverted epifluorescence microscope with a 60× objective and GFP/DsRed filter sets and an ICAM camera. The 2P-FLIM images were collected using the TCSPC fast scanning imaging mode as described under Methods. (a) Conventional epifluorescence image of GFP-PKC distribution in a resting CHO cell, (b) lifetime image of the same cell with the analysis area enclosed by the red line (cytosol) shown (with colour coding) in the inset and giving an average lifetime of ~2.2 ns and (c) with the analysis area enclosed by the red line (nucleus) shown in the inset giving an average lifetime of ~2.0 ns. Cells shown are representative images from replicate experiments.

Mentions: A lifetime image of a CHO cell transiently transfected with GFP-PKCα for 48 hr is shown in Figure 2, with the corresponding epifluorescence image. It can be seen that PKCα is widely and abundantly distributed in the cell cytoplasm and nucleus. The data collection time for this and other lifetime images was optimally 2–3 minutes. The lifetime data for the various conditions examined is also summarised in Table 1.


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)

Fluorescence lifetime imaging of GFP-PKC expressed in CHO cells. GFP-PKC was transiently expressed in CHO cells as described in the legend in Figure 1 and detailed under Methods. Images were acquired using a Nikon 2000 inverted epifluorescence microscope with a 60× objective and GFP/DsRed filter sets and an ICAM camera. The 2P-FLIM images were collected using the TCSPC fast scanning imaging mode as described under Methods. (a) Conventional epifluorescence image of GFP-PKC distribution in a resting CHO cell, (b) lifetime image of the same cell with the analysis area enclosed by the red line (cytosol) shown (with colour coding) in the inset and giving an average lifetime of ~2.2 ns and (c) with the analysis area enclosed by the red line (nucleus) shown in the inset giving an average lifetime of ~2.0 ns. Cells shown are representative images from replicate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1131895&req=5

Figure 2: Fluorescence lifetime imaging of GFP-PKC expressed in CHO cells. GFP-PKC was transiently expressed in CHO cells as described in the legend in Figure 1 and detailed under Methods. Images were acquired using a Nikon 2000 inverted epifluorescence microscope with a 60× objective and GFP/DsRed filter sets and an ICAM camera. The 2P-FLIM images were collected using the TCSPC fast scanning imaging mode as described under Methods. (a) Conventional epifluorescence image of GFP-PKC distribution in a resting CHO cell, (b) lifetime image of the same cell with the analysis area enclosed by the red line (cytosol) shown (with colour coding) in the inset and giving an average lifetime of ~2.2 ns and (c) with the analysis area enclosed by the red line (nucleus) shown in the inset giving an average lifetime of ~2.0 ns. Cells shown are representative images from replicate experiments.
Mentions: A lifetime image of a CHO cell transiently transfected with GFP-PKCα for 48 hr is shown in Figure 2, with the corresponding epifluorescence image. It can be seen that PKCα is widely and abundantly distributed in the cell cytoplasm and nucleus. The data collection time for this and other lifetime images was optimally 2–3 minutes. The lifetime data for the various conditions examined is also summarised in Table 1.

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