The segregation of eukaryotic chromosomes during mitosis requires their extensive folding

The segregation of eukaryotic chromosomes during mitosis requires their extensive folding into units of manageable size for the mitotic spindle. S10 phosphorylation and deacetylation of histone H4 promote short-range compaction of chromatin during early anaphase. Independently condensin mediates the axial contraction of chromosome arms a process peaking later in anaphase. Whereas defects in chromatin compaction have no observable effect on axial contraction and condensin inactivation does not affect short-range chromatin compaction inactivation of both pathways causes synergistic defects in chromosome segregation and cell viability. Furthermore both pathways Staurosporine rely at least partially around the deacetylase Hst2 suggesting that this protein helps coordinating chromatin compaction and axial contraction to properly shape Staurosporine mitotic chromosomes. DOI: locus on chromosome IV 15 kb from ?mutations all abolished the reduction in brightness of the TetR-mCherry focus normally observed in anaphase cells (Physique 1B). In reverse mutations that promote constitutive H2A/H4 conversation such as and caused the TetR-mCherry focus to constitutively show that is even in G1 cells the low fluorescence intensity normally specific of anaphase cells. The effect of the mutation was indeed mediated by the recruitment of Hst2 since the mutation in the locus or TetO/TetR-mCherry we also measured the fluorescence intensity at the locus in the middle of the right arm of chromosome IV where we integrated LacO repeats in cells expressing LacI fused to Green Fluorescent Protein?(GFP). Although the effect was slightly less pronounced we observed a similar and significant decrease in reporter brightness in anaphase compared with G1 cells at this locus (Physique 1C). As for the locus mutants in the H3 S10 pathway also affected fluctuations in intensity at the CEN distal locus: and gene and the mutation also resulted in a constantly lower fluorescent signal. These data indicated that changes in chromatin business during mitosis indeed affected either the recruitment or the fluorescence Staurosporine intensity of TetR-mCherry and LacI-GFP on two distant chromatin loci one close to the centromere and the second in the middle of the second longest yeast chromosome arm. Since chromatin condensation is usually regulated by the kinase aurora B (Ipl1 in budding yeast) we last asked whether Ipl1 activity is required for the intensity decrease of TetR-mCherry at the locus in mitotic cells. We arrested wild type yeast cells and cells made up of the heat sensitive allele in G1 with alpha-factor released them at the restrictive heat of 35oC and decided TetO/TetR-mCherry fluorescence intensity in the same G1 or following anaphase (Physique 1D). Whereas wild type Gata1 cells showed no significant difference in G1 and anaphase TetO/TetR-mCherry fluorescence Staurosporine intensity the strain showed a significantly brighter dot when undergoing anaphase at the restrictive heat. Compaction in G1 of cells at the restrictive heat was not affected presumably due to the fact that this protein has no activity in G1 even in wild type cells (Buvelot et al. 2003 Thus we conclude that this enhanced H2A-H4 conversation brought on by aurora B-dependent recruitment of the deacetylase Hst2 onto chromatin indeed affects the Staurosporine intensity of the TetR-mCherry signal around the chromosome. Staurosporine Fluorophore concentration quenching causes fluctuations in brightness We next wanted to better understand the molecular processes and structural changes of chromatin that were underlying the fluorescence variation at the TetO array over the cell cycle. Assuming enhanced nucleosome-nucleosome conversation promotes chromatin compaction three models may explain the observed decrease of fluorescence in mitosis. First chromatin compaction might reduce access of DNA-binding proteins such as TetR-mCherry to their binding site on DNA and cause their removal as postulated by the chromosome cleansing hypothesis. Second chromatin compaction might increase the local packing of TetR-mCherry leading to quenching of the fluorophore (Lakowicz 2013 these two first models are depicted in Physique 2A. Third the changed local environment of mitotic chromatin might reduce the intrinsic fluorescence of mCherry and GFP. Physique 2. Fluorophore quenching causes changes of.