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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 and Parker, AW 2005, 'The use of time-resolved fluorescence imaging in the study of protein kinase C localisation in cells' , BMC Cell Biology, 6 (1) , p. 22.

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    Abstract

    Background: Two-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 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-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 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 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. Conclusion: Based 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.

    Item Type: Article
    Themes: Subjects / Themes > Q Science > Q Science (General)
    Subjects / Themes > Q Science > QH Natural history > QH301 Biology
    Subjects outside of the University Themes
    Schools: Colleges and Schools > College of Science & Technology
    Colleges and Schools > College of Science & Technology > School of Environment and Life Sciences
    Journal or Publication Title: BMC Cell Biology
    Publisher: BioMed Central
    Refereed: Yes
    ISSN: 14712121
    Depositing User: H Kenna
    Date Deposited: 10 Aug 2007 14:44
    Last Modified: 20 Aug 2013 16:46
    URI: http://usir.salford.ac.uk/id/eprint/245

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