MADS box transcription factors act in a combinatorial manner either as homo-, heterodimer or higher-order complex and they control floral organ formation and identity and many other developmental processes, through a complex network of protein-protein and protein-DNA interactions. We are studying the behaviour of these proteins in living cells by sophisticated microspectroscopic approaches.
Fluorescence Resonance Energy Transfer (FRET) based methods have become a key for the detection of protein-protein interactions in living cells. In its principle, excited-state energy is transferred non-radiatively through space from a donor to an acceptor molecule.
We have analysed MADS box protein interactions in living cells by means of FRET analyses (See: Immink et al., 2002; Nougalli Tonaco et al., 2006). FRET can be quantified by observing changes in the fluorescence lifetime of the donor, using Fluorescence Lifetime Imaging Microscopy (FLIM). In case of a protein-protein interaction, FRET will occur and the fluorescence lifetime of the donor molecule will decrease. This technique was used to determine interactions between the petunia ovule specific proteins FBP24, FBP2 and FBP11. The experiments were done at the Microspectroscopy Centre in Wageningen.
Localisation of CFP and YFP fused to a MADS box protein. Right: FRET-FLIM lifetime image of a nucleus, showing interaction between two transcription factors in living plant cells.
Selected references
- Immink, R.G.H., Gadella, T.W.J., Ferrario, S.I.T., Busscher, M., and Angenent, G.C. (2002) Analysis of MADS box protein-protein interactions in living plant cells. Proc. Nat. Acad.Sci. USA, 99, 2416-2421.
Immink, R.G.H., and Angenent, G.C. Transcription factors do it together: The hows and whys of studying protein-protein interactions. (2002). Trends in Plant Sciences, 7, 531-534.- Immink, R.G.H., Ferrario, S., Busscher-Lange, J., Kooiker, M., Busscher, M., and Angenent, G.C. (2003) Analysis of the petunia MADS box transcription factor family. Mol Genet Genomics 268: 598-606.
- Nougalli Tonaco, I.A., J.W. Borst, S.C. de Vries, G.C. Angenent & R.G.H. Immink. 2006. In vivo imaging of MADS box transcription factor interactions. Journal of Experimental Botany 57: 33-42.
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