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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 co-expressed with DsRed-cav in CHO cells. 2P-FLIM images were collected as described in the legend to Figure 2. Co-expression of the GFP-PKC with DsRed-cav does not affect the lifetime of the GFP showing that in the unstimulated state PKC is not associated with caveolin. Epifluorescence images for excitation of DsRed (a) and GFP along with DsRed (b) showing that the PKC and caveolin co-distributed in the cytosol. Fluorescence lifetime images with the analysis area enclosed by the red line, (cytosol) (c) or nucleus both essentially showing a lifetime as for Figs 2–3 centred around ~2.2 ns. Cells shown are representative images from replicate experiments.
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Figure 4: Fluorescence lifetime imaging of GFP-PKC co-expressed with DsRed-cav in CHO cells. 2P-FLIM images were collected as described in the legend to Figure 2. Co-expression of the GFP-PKC with DsRed-cav does not affect the lifetime of the GFP showing that in the unstimulated state PKC is not associated with caveolin. Epifluorescence images for excitation of DsRed (a) and GFP along with DsRed (b) showing that the PKC and caveolin co-distributed in the cytosol. Fluorescence lifetime images with the analysis area enclosed by the red line, (cytosol) (c) or nucleus both essentially showing a lifetime as for Figs 2–3 centred around ~2.2 ns. Cells shown are representative images from replicate experiments.

Mentions: The images in Figure 4 are from cells with GFP-PKC co-expressed with DsRed-cav. The epifluorescence images show that both PKC and caveolin were widely distributed in most cell areas, except that caveolin was not found in the nucleus and was concentrated in the perinuclear region. The lifetime images of GFP-PKC were acquired under conditions that would produce no contributions from DsRed fluorescence. This was achieved since the 2-P excitation was nominally at 425 nm, where there is virtually no excitation of the DsRed, also a 500 nm band-pass filter was used to exclude DsRed emission. Therefore only the GFP emission was collected. If the DsRed were to be close enough to PKC then a shortened lifetime would result due to FRET occurring. Again the average lifetime of the GFP-PKC was >2 ns, showing that under conditions when the cell is not stimulated (as defined here by Ca2+-mobilisation or phorbol ester activation of PKC), then PKC and caveolin do not co-associate in the cell.


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 co-expressed with DsRed-cav in CHO cells. 2P-FLIM images were collected as described in the legend to Figure 2. Co-expression of the GFP-PKC with DsRed-cav does not affect the lifetime of the GFP showing that in the unstimulated state PKC is not associated with caveolin. Epifluorescence images for excitation of DsRed (a) and GFP along with DsRed (b) showing that the PKC and caveolin co-distributed in the cytosol. Fluorescence lifetime images with the analysis area enclosed by the red line, (cytosol) (c) or nucleus both essentially showing a lifetime as for Figs 2–3 centred around ~2.2 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 4: Fluorescence lifetime imaging of GFP-PKC co-expressed with DsRed-cav in CHO cells. 2P-FLIM images were collected as described in the legend to Figure 2. Co-expression of the GFP-PKC with DsRed-cav does not affect the lifetime of the GFP showing that in the unstimulated state PKC is not associated with caveolin. Epifluorescence images for excitation of DsRed (a) and GFP along with DsRed (b) showing that the PKC and caveolin co-distributed in the cytosol. Fluorescence lifetime images with the analysis area enclosed by the red line, (cytosol) (c) or nucleus both essentially showing a lifetime as for Figs 2–3 centred around ~2.2 ns. Cells shown are representative images from replicate experiments.
Mentions: The images in Figure 4 are from cells with GFP-PKC co-expressed with DsRed-cav. The epifluorescence images show that both PKC and caveolin were widely distributed in most cell areas, except that caveolin was not found in the nucleus and was concentrated in the perinuclear region. The lifetime images of GFP-PKC were acquired under conditions that would produce no contributions from DsRed fluorescence. This was achieved since the 2-P excitation was nominally at 425 nm, where there is virtually no excitation of the DsRed, also a 500 nm band-pass filter was used to exclude DsRed emission. Therefore only the GFP emission was collected. If the DsRed were to be close enough to PKC then a shortened lifetime would result due to FRET occurring. Again the average lifetime of the GFP-PKC was >2 ns, showing that under conditions when the cell is not stimulated (as defined here by Ca2+-mobilisation or phorbol ester activation of PKC), then PKC and caveolin do not co-associate in the cell.

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