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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 Ca2+-ionophore. 2P-FLIM images were collected as described in the legend to Figure 2. Cells were treated with ionophore for 3 min before mounting and fixation as described in Methods. The epifluorescence image in the inset shows the DsRed-cav distribution (cytoplasmic) which was not affected by the Ca2+ ionophore. When the cytoplasmic area was analysed, (a) as shown by the area within the red line, both orange and green/blue areas are seen indicating the presence of both GFP-PKC and quenched GFP-PKC – note that only GFP lifetime can be observed in the lifetime images. This indicates that DsRed-cav was sufficiently close to the PKC-GFP to induce a quenching of the GFP by the DsRed, i.e. the PKC is translocating to caveolin containing areas. By contrast, in the nucleus (b) only GFP-PKC was expressed and the lifetime was unquenched (~2.2 ns). This is the same as the lifetime for GFP-PKC when only the latter is expressed (see Figure 2). Cells shown are representative images from replicate experiments.
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Figure 5: Lifetime imaging of GFP-PKC co-expressed with DsRed-cav in CHO cells: effect of Ca2+-ionophore. 2P-FLIM images were collected as described in the legend to Figure 2. Cells were treated with ionophore for 3 min before mounting and fixation as described in Methods. The epifluorescence image in the inset shows the DsRed-cav distribution (cytoplasmic) which was not affected by the Ca2+ ionophore. When the cytoplasmic area was analysed, (a) as shown by the area within the red line, both orange and green/blue areas are seen indicating the presence of both GFP-PKC and quenched GFP-PKC – note that only GFP lifetime can be observed in the lifetime images. This indicates that DsRed-cav was sufficiently close to the PKC-GFP to induce a quenching of the GFP by the DsRed, i.e. the PKC is translocating to caveolin containing areas. By contrast, in the nucleus (b) only GFP-PKC was expressed and the lifetime was unquenched (~2.2 ns). This is the same as the lifetime for GFP-PKC when only the latter is expressed (see Figure 2). Cells shown are representative images from replicate experiments.

Mentions: When Ca2+ ionophore is added to cells Ca2+ gains entry and this induces immediate PKC translocation to various locations within the cells, including the perinuclear regions and peripheral membranes, as has been extensively shown in the literature (e.g. see ref [28,16,29]). The localisation of caveolin did not appear to change upon addition of Ca2+ ionophore to the cells (results not shown). For lifetime images it is important to note that what we are observing is the lifetime of the GFP, not its intensity. The lifetime images shown in Figure 5 show that the GFP-PKC fluorescence lifetime in the cytoplasm is reduced to ~1.6 ns. This is due to the quenching by DsRed, since the lifetime is unaffected in the absence of DsRed-cav. By contrast, the GFP-PKC lifetime in the nucleus was unaffected by the Ca2+ treatment, since there was little or no caveolin within the nucleus (Figure 5).


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 Ca2+-ionophore. 2P-FLIM images were collected as described in the legend to Figure 2. Cells were treated with ionophore for 3 min before mounting and fixation as described in Methods. The epifluorescence image in the inset shows the DsRed-cav distribution (cytoplasmic) which was not affected by the Ca2+ ionophore. When the cytoplasmic area was analysed, (a) as shown by the area within the red line, both orange and green/blue areas are seen indicating the presence of both GFP-PKC and quenched GFP-PKC – note that only GFP lifetime can be observed in the lifetime images. This indicates that DsRed-cav was sufficiently close to the PKC-GFP to induce a quenching of the GFP by the DsRed, i.e. the PKC is translocating to caveolin containing areas. By contrast, in the nucleus (b) only GFP-PKC was expressed and the lifetime was unquenched (~2.2 ns). This is the same as the lifetime for GFP-PKC when only the latter is expressed (see Figure 2). Cells shown are representative images from replicate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 5: Lifetime imaging of GFP-PKC co-expressed with DsRed-cav in CHO cells: effect of Ca2+-ionophore. 2P-FLIM images were collected as described in the legend to Figure 2. Cells were treated with ionophore for 3 min before mounting and fixation as described in Methods. The epifluorescence image in the inset shows the DsRed-cav distribution (cytoplasmic) which was not affected by the Ca2+ ionophore. When the cytoplasmic area was analysed, (a) as shown by the area within the red line, both orange and green/blue areas are seen indicating the presence of both GFP-PKC and quenched GFP-PKC – note that only GFP lifetime can be observed in the lifetime images. This indicates that DsRed-cav was sufficiently close to the PKC-GFP to induce a quenching of the GFP by the DsRed, i.e. the PKC is translocating to caveolin containing areas. By contrast, in the nucleus (b) only GFP-PKC was expressed and the lifetime was unquenched (~2.2 ns). This is the same as the lifetime for GFP-PKC when only the latter is expressed (see Figure 2). Cells shown are representative images from replicate experiments.
Mentions: When Ca2+ ionophore is added to cells Ca2+ gains entry and this induces immediate PKC translocation to various locations within the cells, including the perinuclear regions and peripheral membranes, as has been extensively shown in the literature (e.g. see ref [28,16,29]). The localisation of caveolin did not appear to change upon addition of Ca2+ ionophore to the cells (results not shown). For lifetime images it is important to note that what we are observing is the lifetime of the GFP, not its intensity. The lifetime images shown in Figure 5 show that the GFP-PKC fluorescence lifetime in the cytoplasm is reduced to ~1.6 ns. This is due to the quenching by DsRed, since the lifetime is unaffected in the absence of DsRed-cav. By contrast, the GFP-PKC lifetime in the nucleus was unaffected by the Ca2+ treatment, since there was little or no caveolin within the nucleus (Figure 5).

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