The use of time-resolved fluorescence imaging in the study of protein kinase C localisation in cellsReport as inadecuate




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BMC Cell Biology

, 6:22

First Online: 26 April 2005Received: 24 December 2004Accepted: 26 April 2005

Abstract

BackgroundTwo-photon-excitation fluorescence lifetime imaging 2P-FLIM was used to investigate the association of protein kinase C alpha PKCα with caveolin in CHO cells. PKCα is found widely in the cytoplasm and nucleus in most cells. Upon activation, as a result of increased intracellular Ca 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

ResultsFluorescence energy transfer FRET, between GFP-PKCα 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 ~2.2 ns to ~1.5 ns when the GFP and DsRed were sufficiently close for FRET to occur. This only occurred when intracellular Ca 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 PKCα that was not associated with caveolin in the nucleus and cytoplasm, PKCα 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.

ConclusionBased on the extent of lifetime quenching observed, the results are consistent with a direct interaction between PKCα 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.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2121-6-22 contains supplementary material, which is available to authorized users.

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Author: Christopher D Stubbs - Stanley W Botchway - Simon J Slater - Anthony W Parker

Source: https://link.springer.com/article/10.1186/1471-2121-6-22



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