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)

Methods Mol Biol. 2022:2502:151-160. doi: 10.1007/978-1-0716-2337-4_10.

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.

Keywords: Chromatin; Fluorescence ratiometric; Live imaging; Nuclear membrane; Nuclear pore complex.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Nucleus / metabolism
  • Chromatin* / genetics
  • Chromatin* / metabolism
  • Fluorescence
  • HeLa Cells
  • Heterochromatin / metabolism
  • Histones / metabolism
  • Humans
  • Lamin Type A / genetics
  • Lamin Type A / metabolism
  • Nuclear Envelope* / metabolism
  • Nuclear Pore / metabolism

Substances

  • Chromatin
  • Heterochromatin
  • Histones
  • Lamin Type A