Mapping Absolute DNA Density in Cell Nuclei using Single-molecule Localization Microscopy

Summary

The present protocol describes a method measuring absolute DNA densities within adherent cell nuclei using Voronoi tessellation of single-molecule localization microscopy data, known volume, genome size, and cell cycle stage.

Abstract

Within the cell nucleus, silent genes are generally located in chromatin areas of high density called heterochromatin, whereas active genes can be mostly found at the interface between chromatin and the interchromatin space called euchromatin. At present, the characterization of eu- and heterochromatin is mostly based on epigenetic modifications to histone proteins along the DNA sequence, while little is known about absolute DNA densities across the cell nucleus and their functional implications. Diagrams of the nucleus solely based on biochemical data and assumptions about the nature of chromatin as a polymer differ fundamentally from imaging data generated by high-resolution microscopy. This indicates that these methods are not well-suited for measuring density relationships in situ. We believe that spatial constraints might be involved in gene regulation and have therefore developed a method that allows the measurement of absolute DNA densities in mammalian cell nuclei by transforming super-resolution localization data into true-to-scale density maps by Voronoi tessellation.

Introduction

Since the early days of cell biology, the cell nucleus-the seat of genetic information-has fascinated biologists. After applying self-developed cytological staining methods, Emil Heitz discovered, in 1928, differently, intensely stained areas within the cell nucleus¹. He called the more intensely stained, dense areas "heterochromatin", whereas he named the less strongly stained, less dense areas "euchromatin". Over time, it became apparent that active genes are mostly located in euchromatin, while denser areas were found to be rich in repetitive elements and silent genes. The terms heterochromatin and euchromatin have survived t....

Protocol

NOTE: Figure 3 gives an overview of the workflow described in this section. See the Table of Materials for details related to the reagents, materials, equipment, and software used in this protocol. The code used for this publication can be viewed and downloaded here: https://github.com/irradiator/Mapping-absolute-DNA-density-in-cell-nuclei-using-SMLM-microscopy.

1. Cell culture

1. Cultivate C3H10T1/2, HFB, and HeLa c.......

Discussion

This article outlines how to measure absolute DNA densities in mammalian cell nuclei using SMLM. In addition, we have demonstrated how to determine the cell cycle stage of cultured cells and how to use this information to estimate the amount of DNA present in a light optical ultra-thin section. Also described in detail are the preparation of adherent cells for fBALM SMLM microscopy and how to process SMLM data to generate super-resolved microscopic images of genomic DNA in cell nuclei. Finally, the protocol shows how the.......

Materials

Name	Company	Catalog Number	Comments
Cell Culture
µ-Dish 35 mm, high Grid-500 Glass Bottom	Ibidi	81168
C3H 10T1/2	IMB (Niehrs Lab)
DMEM	ThermoFisher	12320032
dPBS	ThermoFisher	14190144
FBS	Life Technologies	16000-044
HeLa	Microscopy Core Facility (IMB)
HFB	IMB (Kubben Lab)
L-Glutamine	Sigma-Aldrich	G7513
Nonessential Aminoacids and vitamins for HFB
Sodium Pyruvate	S8636
Sample Prep
Catalase	Merck	2593710
Glucose	ThermoFisher	241922500
Glucose Oxidase	Merck	49180
Paraformaldehyde	Sigma-Aldrich	158127
RNase Cocktail	ThermoFisher	AM2286
SYTOX Orange	ThermoFisher	S11368
TetraSpeck Fluorescent Microspheres Sampler Kit	ThermoFisher	T7284
Triton X-100	ThermoFisher	327372500
Software
BioFormats	OpenMicroscopy.org		open source software https://www.openmicroscopy.org/bio-formats/
CellProfiler v4.2.1	CellProfiler.org		open source software https://cellprofiler.org
FiJI	nih.gov		open source software https://imagej.net/software/fiji/?Downloads
LAND	nih.gov		open source software https://github.com/Jan-NM/LAND
MatLab 2021	Math Works		commercial software - requires "Image Processing Toolbox"
R v.4..0.3	r-project.org		open source software https://www.r-project.org
ThunderSTORM v1.3			open source software https://zitmen.github.io/thunderstorm/
Microscopes:
AF 7000	Leica
Leica GSD	Leica
SMLM Microscope	Cremer Lab		custom-built by Dr. S-Y. Chen