Correlating Mobility and Interaction of Transcription Factors by SPIM-FCS

Abstract

In single plane illumination microscopy (SPIM) an image of a two-dimensional slice of a sample is formed by illumination with a laser beam focused in only one dimension. Thus, fluorescence fluctuations may be observed simultaneously in all voxels of this 2D slice, thereby obtaining diffusion and transport coefficients as well as concentrations of fluorescently labeled biomolecules, creating a “mobility map” of the observed cell.By combining SPIM with fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS), direct information on the strength of molecular interactions can be obtained. We built a dual color SPIM-FCCS with two laser wavelengths for illumination and dual-channel detection through either an EMCCD camera or an ultrafast avalanche photodiode matrix detector, with wavelength splitting optics to image the two colour channels simultaneously. This system offers a time resolution in the range of 105 frames/sec.Here we demonstrate that SPIM-FCS/FCCS possesses the sensitivity to detect and quantify protein-protein interactions in live cells by characterizing the interaction of the subunits of heterodimeric transcription factors, c-Fos/c-Jun and IQGAP/cdc42. The protein-protein interaction clearly shows up in the cross-correlation amplitude. Analysis of the spatial distribution of diffusion coefficients of the fluorescent proteins and of their cross-correlation amplitude shows that formation of the heterodimer is correlated with regions of decreased mobility, probably related to DNA binding.