Preview

PDF, English Download (9MB) | Terms of use

Abstract

The regulation of DNA transcription into RNA and its translation into proteins is one of the most important processes determining cell behaviour. Transcription factors regulate DNA transcription into RNA, thereby controlling protein expression and cellular processes. General transcription factors like c-Fos and c-Jun participate in the regulation of processes such as proliferation, differentiation, apoptosis and oncogenesis. Molecular dynamics inside cells can be investigated by fluorescence correlation spectroscopy (FCS). The fluorescently labeled molecules are excited by a focused laser beam and are characterized by an auto-correlation analysis to yield the information about the diffusion coefficients and concentrations. To study biomolecular interactions, a two-color fluorescence cross-correlation spectroscopy (FCCS) is used. Two molecules are labeled with different fluorophores to distinguish them spectrally. Cross-correlating the fluorescence fluctuation in the detection channels reveals their interaction. These techniques are typically implemented on a confocal microscope, which enables detection of fluorescently labeled molecules with single-molecule sensitivity using the avalanche photodiode detector. A technique that allows imaging in a thin plane and spatially resolves FCCS by analysis of fast image series is single plane illumination microscopy (SPIM). SPIM illuminates the fluorescently labeled sample by a thin light sheet formed by a laser beam focused through cylindrical optics and simultaneously images all points in the two-dimensional field of view with a fast camera. SPIM-FCCS uses two overlapping light sheets of different wavelengths and the two fluorescence channels are optically split to two half planes of the same sensor. In this Thesis, the mobility and interaction of c-Fos and c-Jun transcription factors were studied and characterised in vivo using SPIM-FCCS. I collected mobility and interaction maps of c-Fos-eGFP and c-Jun-mRFP1 transcription factors within living cell nuclei. The c-Fos dimerizes with c-Jun to form the transcription activator protein-1 (AP-1) which binds to the specific recognition site. SPIM-FCCS monitors these processes, which provides diffusion coefficient and protein-protein interaction data in the whole image plane simultaneously, instead of just at one point on conventional confocal FCS. I find a strong correlation between diffusional mobility and interaction: regions of strong interaction show slow mobility. Controls containing an eGFP-mRFP dimer, eGFP and mRFP monomers, or c-Fos-eGFP and c-Jun-mRFP1 mutants lacking dimerization and DNA-binding domains, showed no such correlation.