In common with other signalling proteins, PKC has been suggested to associate with caveolin-1 [1, 2], a key component of caveoli and membrane rafts, that are proposed to exist as signalling platforms in the plasma membrane and elsewhere in the cell. However, where in the cell the PKC-caveolin interaction might occur and under what conditions remains unclear.
Caveolae are vesicular organelles are that are involved in a wide range of cellular functions, serving as platforms or rafts, wherein reside a wide variety of signalling molecules . The caveolin proteins (caveolin-1, -2, and -3) act as the structural components of caveolae. They also function as scaffolding proteins and as such recruit numerous signalling molecules to caveolae where their activity is regulated. PKC is a signalling molecule of major importance in cells, which in the form of twelve isoforms, regulates numerous signalling cascades by virtue of its ability to phosphorylate target proteins that include receptors, G-proteins, ion channels as well as other kinases [4–6]. This leads to control of numerous cellular processes, such as secretion, proliferation, differentiation, apoptosis, permeability, migration, hypertrophy etc [4, 5, 7–11].
While it has been shown that isolated caveoli interact with purified PKCα , PKCγ  and PKCε  using immunoprecipitation, where in the cell this occurs is not known. Caveolin contains a sequence that is a consensus site for phosphorylation by PKC , while down-regulation of plasma membrane-translocated PKCα involvesinternalization of the active enzyme that involves ubiquitination, through a caveolae-dependent mechanism, followed by multisite dephosphorylation and down-regulation in a perinuclear compartment in a time dependent manner (~30 min after stimulated translocation to the plasma membrane) . It has also been shown that endocytic trafficking via caveolae may be a PKCα-dependent process . These observations lead to the question of whether PKCα interacts directly with caveolin, and where in the cell this occurs, a question we examined in this present study.
The classic biochemical or immunoprecipitation approaches for determining the location of signalling molecules in cells, while commonly used, is severely limited for several reasons. The main drawback is that it involves destruction of the cell, resulting in loss of spatial information. Staining the cells with fluorescent antibodies provides a useful advance enabling apparent "co-localisation" to be obtained when two different fluorophores are used. However, the two fluorophores may still be a considerable distance apart without any protein-protein interaction between the pair occurring. The method also suffers from problems of photobleaching and when the probes are in abundance in the cell false co-localisation data can result.
Recently, with the advent of green fluorescent protein (GFP) technology, considerable new information has become available using imaging approaches. This allows the protein of interest to be tagged by expression in the cell, allowing it to be functionally located within the cell without recourse to antibodies. Numerous papers have shown signalling molecules in cells can be tracked as they move between subcellular locations using expression of GFP-tagged proteins and fluorescence microscopy. PKC has been studied in a wide range of situations using GFP-tags (e.g. [18–20]. Lifetime imaging has been used as a localisation tool for GFP-tagged proteins [21–23] and using this approach both PKCα activation levels, along with localisation, has been detected through the binding of fluorescently tagged phosphorylation site-specific antibodies using fluorescence energy transfer (FRET), measured through a donor fluorophore on the PKC . In most cases the localisation was either followed in real-time or after fixation, following initiation of intracellular signalling. There are now a number of different fluorescent proteins available in the "GFP" family, for example cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), red fluorescent protein (DsRed) etc.
A considerable advance over the co-localisation approach is to use steady state FRET between suitably tagged proteins. A further improvement over steady state FRET is achieved by monitoring the lifetime of the donor, which is independent of changes in concentration, photobleaching and various limitations over intensity-based detection. Donor lifetime quenching is evidence for a direct physical interaction and in addition does not require corrections due to spectral over-lap that are required in steady state FRET. The use of this approach FRET-FLIM for localisation using different GFP-type constructs has been described recently in the literature [25–27]. This approach in combination with 2-photon (2P) excitation provides a powerful imaging technique. It does not require complex corrections that intensity-based FRET entails, allows excitation within a narrow focussed plane without contributions outside this region, as would be the case in one-photon excitation, as well as other advantages, such as the ability to image deeper in tissue etc.
In this work the interaction of PKCα with caveolin, was investigated using two-photon-fluorescence lifetime imaging (2P-FLIM). This was determined by investigating the FRET between GFP-tagged PKCα and DsRed-caveolin (DsRed-cav). Using the quenched lifetime of the GFP-tag, areas showing co-localisation in CHO cells was identified after activation of the PKC had occurred. When the GFP-PKC is induced to translocate to membranes, excitation of the GFP at 850 nm, for 2P excitation, should lead to FRET to the DsRed construct if the two are co-localised. This would be seen as a reduced GFP lifetime, in contrast to areas in which the GFP-PKC resides but not with caveolin. This information was determined using a FLIM time-correlated single photon counting set up (TCSPC) with the frequency doubled output of a Tsunami pulsed laser (110 fs), coupled to an inverted microscope. Based on the lifetime quenching data the results were consistent with three populations of activated GFP-PKC. One within the cytoplasmic area of the cells, which was probably undergoing a direct interaction with caveolin, likely in endosomes, a second population in the cytoplasm and in the nucleus that was not interacting with caveolin and a third at the plasma membrane possibly indirectly interacting with caveolin.