Poster Presentation 2014 International Biophysics Congress

Structure(d) illumination - using high resolution structured illumination microscopy for detailed analysis of replication pattern dynamics (#419)

Felix Kraus 1 , Alexei Budco 1 , Yolanda Markaki 1 , Heinrich Leonhardt 1
  1. Ludwig-Maximilians Universität München, Munich-Martinsried, Germany

Accurate and controlled DNA replication during S phase is pivotal for cellular genomic integrity and proliferation.

Initiation and progression of S phase is tightly regulated and spatiotemporally organized in a non-random manner1,2. Gene-rich regions (euchromatin) replicate first (early-S phase), whereas facultative and constitutive heterochromatin replicate in mid and late-S phase, respectively.

Although S phase progression and regulation have been extensively studied and knowledge on the mechanisms is established at the macromolecular level, still information on the organization and structure of replicons (replicated chromatin) is lacking.  

3D-Structured-Illumination-Microscopy (3D-SIM) enabled the visualization of chromatin structural changes/conformation previously not assessable due to the diffraction limit of conventional fluorescence microscopy3.

Combined with optimized bioinformatics solutions for analyzing multidimensional data sets we established an evaluation pipeline for quantifying spatial properties of replication foci (RF). 

We were able to analyze volume differences of newly synthesized DNA compared to compacted already replicated chromatin.  In addition, with the use of a precisely tailored voxel segmentation of DNA labeling intensities reflecting compaction4,5, we mapped RF onto chromatin classes at different S phase stages (early, mid, late).

This work showed for the first time super-resolved active molecular processes occurring at spatially-defined, less compacted chromatin regions, regardless of the original compaction state of these regions (euchromatin, heterochromatin).

This approach could potentially be applied for the study of a wide-range of active processes, such as transcription or DNA repair.

  1. Chagin, Vadim O., Jeffrey H. Stear, and M. Cristina Cardoso. "Organization of DNA replication." Cold Spring Harbor perspectives in biology 2, no. 4 (2010): a000737.
  2. Casas-Delucchi, Corella S., and M. Cristina Cardoso. "Epigenetic control of DNA replication dynamics in mammals." Nucleus 2, no. 5 (2011): 370-382.
  3. Schermelleh, Lothar, Peter M. Carlton, Sebastian Haase, Lin Shao, Lukman Winoto, Peter Kner, Brian Burke et al. "Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy." Science 320, no. 5881 (2008): 1332-1336.
  4. Markaki, Yolanda, Daniel Smeets, Susanne Fiedler, Volker J. Schmid, Lothar Schermelleh, Thomas Cremer, and Marion Cremer. "The potential of 3D‐FISH and super‐resolution structured illumination microscopy for studies of 3D nuclear architecture." Bioessays 34, no. 5 (2012): 412-426.
  5. Smeets, Daniel, Yolanda Markaki, Volker J. Schmid, Felix Kraus, Anna Tattermusch, Andrea Cerase, Michael Sterr et al. "Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci." Epigenetics & Chromatin 7, no. 1 (2014): 8