9. When the root is placed on the slide there is a change of
orientation (from vertical growth to horizontal); as such the
root will adapt to the new gravity direction. To minimize root
movement during the acquisition, the sample can be mounted
and positioned horizontally for 5-10 min prior to imaging.
10. The number of nuclei per acquisition will differ in function of
the root and area of interest. To keep a robust analysis, acquisitions should be taken in the same root area (i.e., using nuclei at
the same stage of development).
11. The laser intensity should be kept as low as possible to avoid
bleaching during the time of the acquisition. If the signal is
weak, the pinhole can be slightly opened to increase the signal.
The number of Z steps and time points can be modified
depending on the samples. For instance, bigger nuclei will
require larger Z-stacks. For our analysis, three Z-steps were
enough to track spots in the nucleus without increasing the
time interval.
12. If the trajectory proposed by the plugin does not correspond to
the actual trajectory of the spot, it is possible to correct the
trajectories manually. To this end, right-click on the image and
click on “Add a node at” or “Remove a node at,” to refine the
trajectory.
13. For more details about the calculation of the radius of constraint see supplementary materials in Neumann et al. [30].
Acknowledgments
We would like to thank Anaı ¨s Cheblal and Susan M. Gasser for
providing an Excel macro for MSD calculation and the SpotTracking2D PlugIn. We are also thankful to Antonius Matzke for
providing the lacO/LacI Arabidopsis lines. This work was supported by Swedish Research Council (Vetenskapsra ˚det) grant number 2018-04101.
References
1. Bannister AJ, Kouzarides T (2011) Regulation
of chromatin by histone modifications. Cell
Res 21:381–395. https://doi.org/10.1038/
cr.2011.22
2. Rosa S, Shaw P (2013) Insights into chromatin
structure and dynamics in plants. Biology
(Basel) 2:1378–1410. https://doi.org/10.
3390/biology2041378
3. Vergara Z, Gutierrez C (2017) Emerging roles
of chromatin in the maintenance of genome
organization and function in plants. Genome
Biol 18:1–12. https://doi.org/10.1186/
s13059-017-1236-9
4. Gu B, Swigut T, Spencley A et al (2018)
Transcription-coupled changes in nuclear
mobility of mammalian cis-regulatory elements. Science 359:1050–1055. https://doi.
org/10.1126/science.aao3136
5. Kimura H (2005) Histone dynamics in living
cells revealed by photobleaching. DNA Repair
(Amst)
4:939–950.
https://doi.org/10.
1016/j.dnarep.2005.04.012
222
Anis Meschichi and Stefanie Rosa
orientation (from vertical growth to horizontal); as such the
root will adapt to the new gravity direction. To minimize root
movement during the acquisition, the sample can be mounted
and positioned horizontally for 5-10 min prior to imaging.
10. The number of nuclei per acquisition will differ in function of
the root and area of interest. To keep a robust analysis, acquisitions should be taken in the same root area (i.e., using nuclei at
the same stage of development).
11. The laser intensity should be kept as low as possible to avoid
bleaching during the time of the acquisition. If the signal is
weak, the pinhole can be slightly opened to increase the signal.
The number of Z steps and time points can be modified
depending on the samples. For instance, bigger nuclei will
require larger Z-stacks. For our analysis, three Z-steps were
enough to track spots in the nucleus without increasing the
time interval.
12. If the trajectory proposed by the plugin does not correspond to
the actual trajectory of the spot, it is possible to correct the
trajectories manually. To this end, right-click on the image and
click on “Add a node at” or “Remove a node at,” to refine the
trajectory.
13. For more details about the calculation of the radius of constraint see supplementary materials in Neumann et al. [30].
Acknowledgments
We would like to thank Anaı ¨s Cheblal and Susan M. Gasser for
providing an Excel macro for MSD calculation and the SpotTracking2D PlugIn. We are also thankful to Antonius Matzke for
providing the lacO/LacI Arabidopsis lines. This work was supported by Swedish Research Council (Vetenskapsra ˚det) grant number 2018-04101.
References
1. Bannister AJ, Kouzarides T (2011) Regulation
of chromatin by histone modifications. Cell
Res 21:381–395. https://doi.org/10.1038/
cr.2011.22
2. Rosa S, Shaw P (2013) Insights into chromatin
structure and dynamics in plants. Biology
(Basel) 2:1378–1410. https://doi.org/10.
3390/biology2041378
3. Vergara Z, Gutierrez C (2017) Emerging roles
of chromatin in the maintenance of genome
organization and function in plants. Genome
Biol 18:1–12. https://doi.org/10.1186/
s13059-017-1236-9
4. Gu B, Swigut T, Spencley A et al (2018)
Transcription-coupled changes in nuclear
mobility of mammalian cis-regulatory elements. Science 359:1050–1055. https://doi.
org/10.1126/science.aao3136
5. Kimura H (2005) Histone dynamics in living
cells revealed by photobleaching. DNA Repair
(Amst)
4:939–950.
https://doi.org/10.
1016/j.dnarep.2005.04.012
222
Anis Meschichi and Stefanie Rosa