Fine structure of the "PcG body" in human U-2 OS cells established by correlative light-electron microscopy

Abstract

Polycomb group (PcG) proteins of the Polycomb repressive complex 1 (PRC1) are found to be diffusely distributed in nuclei of cells from various species. However they can also be localized in intensely fluorescent foci, whether imaged using GFP fusions to proteins of PRC1 complex, or by conventional immunofluorescence microscopy. Such foci are termed PcG bodies, and are believed to be situated in the nuclear intechromatin compartment. However, an ultrastructural description of the PcG body has not been reported to date. To establish the ultrastructure of PcG bodies in human U-2 OS cells stably expressing recombinant polycomb BMI1-GFP protein, we used correlative light-electron microscopy (CLEM) implemented with high-pressure freezing, cryosubstitution and on-section labeling of BMI1 protein with immunogold. This approach allowed us to clearly identify fluorescent PcG bodies, not as distinct nuclear bodies, but as nuclear domains enriched in separated heterochromatin fascicles. Importantly, high-pressure freezing and cryosubstitution allowed for a high and clear-cut immunogold BMI1 labeling of heterochromatin structures throughout the nucleus. The density of immunogold labeled BMI1 in the heterochromatin fascicles corresponding to fluorescent "PcG bodies" did not differ from the density of labeling of heterochromatin fascicles outside of the "PcG bodies". Accordingly, an appearance of the fluorescent "PcG bodies" seems to reflect a local accumulation of the labeled heterochromatin structures in the investigated cells. The results of this study should allow expansion of the knowledge about the biological relevance of the "PcG bodies" in human cells.

Nuclear Architecture and Chromatin Dynamics in Interphase Nuclei of <b><i>Arabidopsis thaliana</i></b>

“…Time-lapse experiments also revealed a fast regime of PcG motion within volumes smaller than CTs as well as a slow regime of long-range motion that depended on coordinated large-scale chromatin movements which might be associated with displacements of whole or large parts of chromosomes and led to occasional association and dissociation events of PcG bodies [Cheutin and Cavalli, 2012]. However, whether PcG bodies are true nuclear entities or 3D chromosomal domains enriched in PcG proteins is still a matter of debate [Smigova et al, 2011;Pirrotta and Li, 2012]. Thus, the mechanism by which PcG bodies form, which may involve PRC1 components with polymerization properties [Isono et al, 2013], as well as the role of PcG bodies in transcriptional regulation remain to be clarified.…”

Ultrastructure of Cytoplasmic and Nuclear Inosine-5’-Monophosphate Dehydrogenase 2 “Rods and Rings” Inclusions

“…All of the previous steps were completed at room temperature. Polymerization was carried out at 60C for 72 hr (adapted from Smigova et al 2011). Such prepared samples were sectioned into ~200-nm thin sections and used for electron tomography (see below).…”

“…Final hardening of resin was performed at 20C for 24 hr. Polymerization of Lowicryl resin was conducted under an ultraviolet LED lamp (Leica EM FSP), as described elsewhere (Smigova et al 2011).…”

A simple Fourier filter for suppression of the missing wedge ray artefacts in single-axis electron tomographic reconstructions

“…Samples were processed according to (Smigova et al, 2011). Briefly, human Hep2 cells were treated by IMPDH inhibitor ribavirin.…”