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Imaging Fos-Jun transcription factor mobility and interaction in live cells by single plane illumination-fluorescence cross correlation spectroscopy.

ABSTRACT: We collected mobility and interaction maps of c-Fos-eGFP and c-Jun-mRFP1 transcription factors within living cell nuclei. c-Fos dimerizes with c-Jun to form the transcription activator protein-1 (AP-1) which binds to the specific recognition site. To monitor this process, we used fluorescence cross-correlation spectroscopy on a single plane illumination microscope (SPIM-FCCS), which provides diffusion coefficient and protein-protein interaction data in the whole image plane simultaneously, instead of just one point on conventional confocal FCS. We find a strong correlation between diffusional mobility and interaction: regions of strong interaction show slow mobility. Controls containing either an eGFP-mRFP dimer, separately expressing eGFP and mRPF, or c-Fos-eGFP and c-Jun-mRFP1 mutants lacking dimerization and DNA-binding domains, showed no such correlation. These results extend our earlier findings from confocal FCCS to include spatial information.

SUBMITTER: Pernus A

PROVIDER: S-EPMC4397054 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Study of the density, spatial distribution, and molecular interactions of receptors on the cell membrane provides the knowledge required to understand cellular behavior and biological functions, as well as to discover, design, and screen novel therapeutic agents. However, the mapping of receptor distribution and the monitoring of ligand-receptor interactions on live cells in a spatially and temporally ordered manner are challenging tasks. In this paper, we apply fluorescence correlation spectroscopy (FCS) to map receptor densities on live cell membranes by introducing fluorescently marked aptamer molecules, which specifically bind to certain cell-surface receptors. The femtoliter-sized (0.4 fL) observation volume created by FCS allows fluorescent-aptamer detection down to 2 molecules and appears to be an ideal and highly sensitive biophysical tool for studying molecular interactions on live cells. Fluorophore-labeled aptamers were chosen for receptor recognition because of their high binding affinity and specificity. Aptamer sgc8, generated for specific cell recognition by a process called cell systematic evolution of ligands by exponential enrichment, was determined by FCS to have a binding affinity in the picomolar range (dissociation constant K(d)=790+/-150 pM) with its target membrane receptor, human protein tyrosine kinase-7 (PTK7), a potential cancer biomarker. We then constructed a cellular model and applied this aptamer-receptor interaction to estimate receptor densities and distributions on the cell surface. Specifically, different expression levels of PTK7 were studied by using human leukemia CCRF-CEM cells (1300+/-190 receptors microm(-2)) and HeLa cervical cancer cells (550+/-90 receptors microm(-2)). Competition studies with excess nonlabeled aptamers and proteinase treatment studies proved the validity of the density-estimation approach. With its intrinsic advantages of direct measurement, high sensitivity, fast analysis, and single-cell measurement, this FCS density-estimation approach holds potential for future applications in molecular-interaction studies and density estimations for subcellular structures and membrane receptors.

Dataset Information

Imaging Fos-Jun transcription factor mobility and interaction in live cells by single plane illumination-fluorescence cross correlation spectroscopy.

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