Analyzing Chromatin Condensation in Yeast by Second Harmonic Generation Microscopy

The DNA in the nucleus is packed by proteins into chromatin fibers. During the cell cycle the interphase chromatin is condensed by members of the Structural Maintenance of Chromosome (SMC) family of protein complexes. Chromosomes reach maximum level of condensation in pro-metaphase. In mammalian cells, condensed mitotic chromosomes are visualized as individual bodies. However, the small size of the nucleus in budding yeast (Saccharomyces cerevisiae) and the low level of condensation make the assessment of condensation in these cells a challenging task. Several methods have been developed to study condensation in yeast. However, all of them suffer from major weaknesses. We developed a new method to study chromatin condensation in live yeast cells that is based on second harmonic generation (SHG) microscopy. SHG is a physical phenomenon of the second order in which the energy of consecutive photons is reflected from isotropic molecules, such as chromatin, One challenge that we encountered in generating second harmonic was the requirement to accurately focus the laser on the cell nucleus.

We utilized this method to analyze changes in chromatin density throughout the cell cycle in yeast. Furthermore, we showed that SMCs play a central role in chromatin organization and mediates condensation. This method provides a new tool to study chromatin structure in live yeast cells.