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


CONCLUSIONS
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.
Stubbs
Christopher D
CD
Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA. cstubbs@stubbsmail.com
Botchway
Stanley W
SW
Slater
Simon J
SJ
Parker
Anthony W
AW
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D022461:Caveolins / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D006207:Half-Life; D010703:Phorbol Esters / Q000494:pharmacology; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D015398:Signal Transduction; D014162:Transfection
D022461:Caveolins; D005456:Fluorescent Dyes; D010703:Phorbol Esters; D051571:Protein Kinase C-alpha; D002118:Calcium