Abstract
The development of progressively sophisticated tools complemented by the integration of live cell imaging enhances our understanding of the four-dimensional (4D) nucleome, revealing elaborate molecular interactions and chromatin states. Yet, the dynamics of chromosomes in relation to nuclear organelles or to each other across cell cycle in living cells are underexplored. We have developed photoconvertible GFP H3-Dendra2 stably expressing in PC3M cells. The nuclear lamina and perinucleolar associated heterochromatin or diffuse chromosome regions were photoconverted through a single-point activation using a confocal microscope. The results demonstrated a dynamic nature for both types of chromosomes in the same cell cycle and across mitosis. While some chromosome domains were heritably associated with either nuclear lamina or nucleoli, others changed alliance to different nuclear organelles postmitotically. In addition, co-photoconverted chromosome domains often do not stay together within the same cell cycle and across mitosis, suggesting a transient nature of chromosome neighborhoods. Long-range spreading and movement of chromosomes were also observed. Interestingly, when cells were treated with a low concentration of actinomycin D that inhibits Pol I transcription through intercalating GC-rich DNA, chromosome movement was significantly blocked. Treatment with another Pol I inhibitor, metarrestin, which does not impact DNA, had little effect on the movement, suggesting that the DNA structure itself plays a role in chromosome dynamics. Furthermore, inhibition of Pol II transcription with α-amanitin also reduced the chromosome movement, demonstrating that Pol II, but not Pol I transcription, is important for chromosome dynamics in the nucleus.
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Acknowledgements
We would like to thank Michael Davidson (University of Florida) for the pcDNA3.1 Dendra2. We acknowledge that imaging work was performed at the Northwestern University Center for Advanced Microscopy, generously supported by NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center. Wei Hong Yeo is supported by the Christina Enroth-Cugell and David Cugell Fellowship for Visual Neuroscience and Biomedical Engineering. We thank the NIH funding for HFZ (R01GM139151, R01GM140478, and U54CA268084) and for SH (U01DA052772).
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L.M.U.P performed cell maintenance and photoconversion experiments. W.H.Y performed image preprocessing and statistical analyses using MATLAB. L.M.U.P. and W.H.Y wrote the Materials and Methods. L.M.U.P. prepared Figs. 1–5 and supplementary videos (ESM_1, ESM_2, ESM_3). W.H.Y prepared Figs. 5d, S1, S2. S.H. wrote the Abstract, Introduction, Results and Discussion, and References. All authors reviewed the manuscript.
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Hao F. Zhang has financial interests in Opticent Inc., which did not support this work. The other authors declare no conflicts of interest.
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Phan, L.M.U., Yeo, WH., Zhang, H.F. et al. Dynamic chromosome association with nuclear organelles in living cells. Histochem Cell Biol 162, 149–159 (2024). https://doi.org/10.1007/s00418-024-02288-8
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DOI: https://doi.org/10.1007/s00418-024-02288-8