Presently, it is thought that native chromatin is distributed uniformly throughout the nucleus. However, their research has in fact shown that native chromatin (both active and nonactive) is located near the nuclear envelope.
In this study, researchers used ZEISS arivis Pro (formerly Vision4D) and a Matryoshka (Russian doll) script to generate concentric spheric volumes that allowed for the measurement of chromatin distribution in live 3D cell volumes.
Video courtesy: Amiad Pavlov et al. 3D view of a live control nucleus showing chromatin at the periphery. The segmented nucleus is divided into 10 concentric 3D radial shells for quantification of radial chromatin distribution.
3D visualization of the live muscle nuclei revealed a peripheral distribution of the chromatin, with a substantial region in the interior of the nucleus that was devoid of chromatin. The nuclear envelope was labeled with nesprin/klar–GFP (green fluorescent protein) and chromatin was labelled by expression of His2B-mRFP (histone H2B–red fluorescent protein).
To quantify chromatin distribution along the radial direction, the researchers segmented each nucleus in 3D and divided into 10 concentric shells. Chromatin density for each shell was calculated from the sum of His2B-mRFP fluorescence intensity, divided by the shell volume.
The nuclear lamina, a thick meshwork of intermediate filaments associated with the inner nuclear membrane, is a major regulator of chromatin architecture, as it tethers mostly dense heterochromatin at specific sequences termed lamina-associated domains (LADs).
LADs are specifically sensitive to the levels of lamin A/C at the nuclear lamina. It was found that peripheral chromatin architecture was sensitive to overexpression (OE) of lamin A/C resulting in chromatin condensation toward the centre of the nucleus.
The study presented the first 3D analysis of global chromatin organization of fully differentiated nuclei within the preserved physiological environment of a live, intact organism. ZEISS arivis Pro (formerly Vision4D) allowed the researchers to create analysis protocols that quantify chromatin contained within the nuclear envelope.
Use of a bespoke Matryoshka script for quantifying the distribution of the chromatin was possible due to the versatility of the software in allowing users to apply their own scripts to their datasets directly in ZEISS arivis Pro (formerly Vision4D).
Image courtesy: Amiad Pavlov et al. 3D view of a live nucleus, overexpressing lamin C, cut through the middle. Chromatin is labeled with His2B-mRFP and nuclear envelope with lamin C–GFP. The segmented nucleus is divided into 10 concentric 3D radial shells (gray) for quantification of radial chromatin distribution.
Challenges and advantages:
This tutorial gives an overview of how to use ZEISS arivis Pro (formerly Vision4D) to run a Matryoshka analysis.
For more information on accessing the Matryoshka Python script, please visit the arivis Knowledge Base.
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