Current models postulate that cohesin is essential for the organization and shaping of the genome into chromatin loops/domains 1. Hi-C data based on large cell populations showed that cohesin loss eliminates all loop domains 2. Here we aimed to investigate the effect of cohesin loss on the preservation/alteration of higher order chromatin structures on the single cell level using super-resolution microscopy and live cell studies. As reference structures we chose replication domains (RDs), first detected in S-phase nuclei as replication foci. RDs stably persist as basic units of higher order chromatin domains (CDs) throughout interphase and during subsequent cell cycles 4. Methods & Results All experiments were performed with HCT-116-RAD21-mAID-mClover cells, where degradation of cohesin is achieved by auxin-induced proteolysis of its subunit RAD21 6. In live cell studies we demonstrated the appearance of mitotic cells up to ~16h after cohesin degradation with a strongly prolongated mitotic phase and final transition into one multilobulated nucleus. Typical RD patterns, achieved by replication-scratch labeling, persisted after cohesin degradation at least up to 46h and were transmitted over mitosis irrespective of highly abnormal nuclear morphologies. No significant difference of RD patterns and size of individual RDs was noted at the resolution level of 3D-SIM between control cells and cells, which were replication-labeled before cohesin degradation. By contrast, when replication-labeling was performed after cohesin degradation, we noted a reduced number and coarsening of RD structures. Conclusions Our microscopic observations provided information on the effects of a cohesin degradation in individual cells, which were not recognized in Hi-C studies of entire cultures. These findings exemplify the importance to combine Hi-C with advanced 3D and 4D microscopy. Single cell Hi-C studies of individual interphase and mitotic cells identified by microscopy could help to further study connections between the level of chromatin loops and higher order chromatin arrangements in interphase and mitosis following cohesin depletion. References: 1 Nishiyama, T. Curr Opin Cell Biol 58, 8-14, doi:10.1016/j.ceb.2018.11.006 (2018). 2 Rao, S. S. P. et al. Cell 171, 305-320 e324, doi:10.1016/j.cell.2017.09.026 (2017). 3 Bintu, B. et al. Science 362, doi:10.1126/science.aau1783 (2018). 4 Cremer, T. et al. Curr Opin Cell Biol 18, 307-316, doi:10.1016/j.ceb.2006.04.007 (2006). 5 Pope, B. D. et al. Nature 515, 402-405, doi:10.1038/nature13986 (2014). 6 Natsume, T. Cell Rep 15, 210-218 (2016).