Chapter 10
Visualizing and Measuring Single Locus Dynamics
in Arabidopsis thaliana
Anis Meschichi and Stefanie Rosa
Abstract
In eukaryotes, DNA is packed into an incredibly complex structure called chromatin. Although chromatin
was often considered as a static entity, it is now clear that chromatin proteins and the chromatin fiber itself
are in fact very dynamic. For instance, the packaging of the DNA into the nucleus requires an extraordinary
degree of compaction but this should be achieved without compromising the accessibility to the transcription machinery and other nuclear processes. Approaches such as gene tagging have been established for
living cells in order to detect, track, and analyze the mobility of single loci. In this chapter, we provide an
experimental protocol for performing locus tracking in Arabidopsis thaliana roots and for characterizing
locus mobility behavior via a Mean Square Displacement analysis.
Key words Locus tagging, Fluorescence microscopy, Green fluorescent protein, Chromatin mobility,
Mean square displacement, Arabidopsis
1 Introduction
Chromatin is composed of DNA wrapped around a complex octamer of proteins called histones, forming the nucleosome. This
structure is highly dynamic due to multiple nuclear processes,
such as transcription, replication, or repair systems, which require
a change in the accessibility of the underlying DNA sequences [1–
3]. Moreover, in recent years, advances on imaging techniques have
started to reveal the dynamic nature of the DNA inside the cell
nucleus [4–7]. Indeed, genes can change their physical location
inside the nucleus with consequences for transcriptional activity
or genome integrity [8–13].
Locus-tagging systems are one approach allowing analyzing the
mobility of a locus through time. There are currently several techniques allowing single locus tagging, such as lacO and tetO system,
or CRISPR-based imaging [14, 15]. The lacO/LacI system is
based on the insertion of lacO arrays in the genome. The lacO
arrays typically consist of 100–256 copies of the lacO sequence
Jose J. Sanchez-Serrano and Julio Salinas (eds.), Arabidopsis Protocols, Methods in Molecular Biology, vol. 2200,
https://doi.org/10.1007/978-1-0716-0880-7_10, © Springer Science+Business Media, LLC, part of Springer Nature 2021
213
Visualizing and Measuring Single Locus Dynamics
in Arabidopsis thaliana
Anis Meschichi and Stefanie Rosa
Abstract
In eukaryotes, DNA is packed into an incredibly complex structure called chromatin. Although chromatin
was often considered as a static entity, it is now clear that chromatin proteins and the chromatin fiber itself
are in fact very dynamic. For instance, the packaging of the DNA into the nucleus requires an extraordinary
degree of compaction but this should be achieved without compromising the accessibility to the transcription machinery and other nuclear processes. Approaches such as gene tagging have been established for
living cells in order to detect, track, and analyze the mobility of single loci. In this chapter, we provide an
experimental protocol for performing locus tracking in Arabidopsis thaliana roots and for characterizing
locus mobility behavior via a Mean Square Displacement analysis.
Key words Locus tagging, Fluorescence microscopy, Green fluorescent protein, Chromatin mobility,
Mean square displacement, Arabidopsis
1 Introduction
Chromatin is composed of DNA wrapped around a complex octamer of proteins called histones, forming the nucleosome. This
structure is highly dynamic due to multiple nuclear processes,
such as transcription, replication, or repair systems, which require
a change in the accessibility of the underlying DNA sequences [1–
3]. Moreover, in recent years, advances on imaging techniques have
started to reveal the dynamic nature of the DNA inside the cell
nucleus [4–7]. Indeed, genes can change their physical location
inside the nucleus with consequences for transcriptional activity
or genome integrity [8–13].
Locus-tagging systems are one approach allowing analyzing the
mobility of a locus through time. There are currently several techniques allowing single locus tagging, such as lacO and tetO system,
or CRISPR-based imaging [14, 15]. The lacO/LacI system is
based on the insertion of lacO arrays in the genome. The lacO
arrays typically consist of 100–256 copies of the lacO sequence
Jose J. Sanchez-Serrano and Julio Salinas (eds.), Arabidopsis Protocols, Methods in Molecular Biology, vol. 2200,
https://doi.org/10.1007/978-1-0716-0880-7_10, © Springer Science+Business Media, LLC, part of Springer Nature 2021
213