Image Scanning Microscopy to Investigate Polycomb Protein Colocalization onto Chromatin

Abstract

Super-resolution microscopy has been recently applied to understand the 3D topology of chromatin at an intermediated genomic scale (kilobases to a few megabases), as this corresponds to a sub-diffraction spatial scale crucial for the regulation of gene transcription. In this context, polycomb proteins are very renowned gene repressors that organize into the multiprotein complexes Polycomb Repressor Complex 1 (PRC1) and 2 (PRC2). PRC1 and PRC2 operate onto the chromatin according to a complex mechanism, which was recently recapitulated into a working model. Here, we present a functional colocalization study at 100–140 nm spatial resolution targeting PRC1 and PRC2 as well as the histone mark H3K27me3 by Image Scanning Microscopy (ISM). ISM offers a more flexible alternative to diffraction-unlimited SRMs such as STORM and STED, and it is perfectly suited to investigate the mesoscale of PRC assembly. Our data suggest a partially simultaneous effort of PRC1 and PRC2 in locally shaping the chromatin topology.

SARS-CoV-2 Infection Alters the Phenotype and Gene Expression of Adipocytes

“…Positive infection of adipocytes was confirmed by confocal imaging: both Spike (Figure 3A, green color) and NC proteins (Figure 3C, yellow color) were detected in infected cells, as compared to control cells not exposed to the virus (Figure 3B,D). Super resolution imaging at mesoscale (100-140 nm) [20] by Image Scanning Microscopy (Airyscan mode) enabled the visualization of single viral particles [21], some of which were found in extremely close proximity to the lipid droplets (LDs) (Figure 3E,F, red Subsequently, we employed the Wuhan strain (B.1) of the coronavirus to infect SGBS cells at PID0 and at PID10, and measured the release of viral particles into the supernatant as a key indicator of productive infection. In Figure 2B, we note a distinctive pattern in the real-time cycle thresholds (Ct) that are inversely related to the abundance of SARS-CoV-2 mRNA.…”

“…Positive infection of adipocytes was confirmed by confocal imaging: both Spike (Figure 3A, green color) and NC proteins (Figure 3C, yellow color) were detected in infected cells, as compared to control cells not exposed to the virus (Figure 3B,D). Super resolution imaging at mesoscale (100-140 nm) [20] by Image Scanning Microscopy (Airyscan mode) enabled the visualization of single viral particles [21], some of which were found in extremely close proximity to the lipid droplets (LDs) (Figure 3E,F, red color). Positive infection of adipocytes was confirmed by confocal imaging: both Spike (Figure 3A, green color) and NC proteins (Figure 3C, yellow color) were detected in infected cells, as compared to control cells not exposed to the virus (Figure 3B,D).…”

On the Advent of Super-Resolution Microscopy in the Realm of Polycomb Proteins

“…To extend the resolution further down to 100–120 nm, ISM usually makes use of an adaptive multi-image deconvolution algorithm. Despite these advantages, ISM has only been minimally applied to investigate the organization of chromatin at sub-diffraction resolution [ 4 ], despite its robust and versatile technical implementation, which affords high spectral multiplexing and may also be compatible with lifetime detection, affording further layers of imaging contrast [ 40 ], although a recent paper by the present authors applied ISM to investigate the functional colocalization of PRC1 and PRC2 in differentiated lung cells [ 45 ].…”

Emerging Mueller matrix microscopy applications in biophysics and biomedicine

“…The SPAD array can be placed in an image plane as it happens for the pinhole. Its sensitive area is comparable to a typical pinhole size (one Airy disk size), and the module can be easily adapted to fit in any confocal microscope [88,91,92].…”