Title : Elastic inter-telomeric tethers and UFBs
Abstract:
Elastic inter-telomeric tethers observed between sister chromatids in mitotic PTK2 cells have raised questions about whether these structures could be ultrafine anaphase DNA bridges (UFBs). To address this, we optimized staining techniques for the Plk1-interacting checkpoint helicase (PICH), known to coat UFBs, and characterized its localization in PTK2 cells. These cells exhibit a small number of large chromosomes and remain very flat during mitosis. We quantified the nuclei size and PICH levels via fluorescence microscopy to optimize our protocols and look for UFBs in mitosis. We put the cells under many different scenarios such as where cells were synchronized using aphidicolin for 17hrs or 2.5 hrs with colcemid and released.
Following drug treatments, cells were either lysed with an extraction PFA solution or lysed and then fixed with 4% PFA in PBS. Cells were immunostained with PICH and DNA was stained with DAPI. Aphidicolin treatment increased PICH staining in the nuclei of PTK2 cells. Of 7,656 cells imaged, we only observed one UFB out of 122 mitotic cells, from the control dish. We also found 559 cells with micro-nuclei(extranuclear bodies formed from chromosomes excluded during division), indicating genomic instability in the current culture, with the control condition showing the highest rate (9.793%). No statistically significant differences in PICH intensity were observed between micronuclei and interphase nuclei. Aphidicolin treatment also led to an increase in cell area, indicating successful cell cycle arrest in S or G2 phases. Prolonged aphidicolin exposure correlated with higher percentages of larger cells, consistent with increased arrest in S or G2, supporting our previous findings. In conclusion, UFBs were hardly present in this population of PTK2. Future studies will include freshly thawed Ptk2 to assess PICH localization compared to this population and to better assess PICH localization in the G1, S, G2, and M phases. We propose to identify the right conditions to increase mitotic after replication stress induction to allow us to better quantify UFBs.
