Monitoring of Chromatin Organization at the Nuclear Pore Complex, Inner Nuclear Membrane, and Nuclear Interior in Live Cells by Fluorescence Ratiometric Imaging of Chromatin (FRIC).

ABSTRACT

The image analysis tool FRIC (Fluorescence Ratiometric Imaging of Chromatin) quantitatively monitors dynamic spatiotemporal distribution of euchromatin and total chromatin in live cells. A vector (pTandemH) assures stoichiometrically constant expression of the histone variants Histone 3.3 and Histone 2B, fused to EGFP and mCherry, respectively. Quantitative ratiometric (H3.3/H2B) imaging displayed a concentrated distribution of heterochromatin in the periphery of U2OS cell nuclei. As a proof of concept, peripheral heterochromatin responded to experimental manipulation of histone acetylation as well as expression of the mutant lamin A protein "progerin," which causes Hutchinson-Gilford Progeria Syndrome. In summary FRIC is versatile, unbiased, robust, requires a minimum of experimental steps and is suitable for screening purposes.