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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 intensity images of GFP-PKC and DsRed-cav expressed in CHO cells. GFP-PKC and/or DsRed-cav were transiently expressed in CHO cells on glass coverslips by culturing for 48 hr. The cells were then treated with Ca2+-ionophore (1 nM) or TPA (100 nM) for 3 min before the cells were fixed on microscope slides as described under Methods. Images were acquired using a Olympus IX-70 inverted epifluorescence microscope with a 60× objective and GFP/DsRed filter sets and a Olympus C3030 camera. (a) a representative image before and (b) after Ca2+-induced translocation by ionophore of GFP-PKC to the peripheral membrane and to discrete regions in the perinuclear region. (c) image of DsRed-cav and (d) GFP-PKC, after treatment with 100 nM TPA.
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Figure 1: Fluorescence intensity images of GFP-PKC and DsRed-cav expressed in CHO cells. GFP-PKC and/or DsRed-cav were transiently expressed in CHO cells on glass coverslips by culturing for 48 hr. The cells were then treated with Ca2+-ionophore (1 nM) or TPA (100 nM) for 3 min before the cells were fixed on microscope slides as described under Methods. Images were acquired using a Olympus IX-70 inverted epifluorescence microscope with a 60× objective and GFP/DsRed filter sets and a Olympus C3030 camera. (a) a representative image before and (b) after Ca2+-induced translocation by ionophore of GFP-PKC to the peripheral membrane and to discrete regions in the perinuclear region. (c) image of DsRed-cav and (d) GFP-PKC, after treatment with 100 nM TPA.

Mentions: The epifluorescence images in Figure 1 show GFP-PKC and DsRed-cav localisation as green and red colours respectively. The images were taken before and after exposure to the phorbol ester TPA for 3 min. It can be seen that PKC accumulated at the cell periphery and in organelles in the perinuclear region.


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 intensity images of GFP-PKC and DsRed-cav expressed in CHO cells. GFP-PKC and/or DsRed-cav were transiently expressed in CHO cells on glass coverslips by culturing for 48 hr. The cells were then treated with Ca2+-ionophore (1 nM) or TPA (100 nM) for 3 min before the cells were fixed on microscope slides as described under Methods. Images were acquired using a Olympus IX-70 inverted epifluorescence microscope with a 60× objective and GFP/DsRed filter sets and a Olympus C3030 camera. (a) a representative image before and (b) after Ca2+-induced translocation by ionophore of GFP-PKC to the peripheral membrane and to discrete regions in the perinuclear region. (c) image of DsRed-cav and (d) GFP-PKC, after treatment with 100 nM TPA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Fluorescence intensity images of GFP-PKC and DsRed-cav expressed in CHO cells. GFP-PKC and/or DsRed-cav were transiently expressed in CHO cells on glass coverslips by culturing for 48 hr. The cells were then treated with Ca2+-ionophore (1 nM) or TPA (100 nM) for 3 min before the cells were fixed on microscope slides as described under Methods. Images were acquired using a Olympus IX-70 inverted epifluorescence microscope with a 60× objective and GFP/DsRed filter sets and a Olympus C3030 camera. (a) a representative image before and (b) after Ca2+-induced translocation by ionophore of GFP-PKC to the peripheral membrane and to discrete regions in the perinuclear region. (c) image of DsRed-cav and (d) GFP-PKC, after treatment with 100 nM TPA.
Mentions: The epifluorescence images in Figure 1 show GFP-PKC and DsRed-cav localisation as green and red colours respectively. The images were taken before and after exposure to the phorbol ester TPA for 3 min. It can be seen that PKC accumulated at the cell periphery and in organelles in the perinuclear region.

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