Department of Cell and Developmental Biology, Northwestern University, Chicago, IL, USA.
Department of Biomedical Engineering, Northwestern University, Evanston, IL, USA.
Histochem Cell Biol. 2024 Jul;162(1-2):149-159. doi: 10.1007/s00418-024-02288-8. Epub 2024 May 30.
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.
不断发展的复杂工具与活细胞成像的整合相辅相成，增强了我们对四维（4D）核组的理解，揭示了精细的分子相互作用和染色质状态。然而，活细胞中染色体与核细胞器或在细胞周期中彼此之间的动态关系仍未得到充分探索。我们开发了可光转化的 GFP H3-Dendra2 在 PC3M 细胞中稳定表达。通过共聚焦显微镜单点激活，对核层和核周异染色质或弥散染色体区域进行光转化。结果表明，在同一细胞周期和有丝分裂过程中，两种类型的染色体都具有动态性质。虽然某些染色体结构域与核层或核仁固有相关，但其他结构域在有丝分裂后会与不同的核细胞器结盟。此外，共同光转化的染色体结构域通常不会在同一个细胞周期内和有丝分裂过程中保持在一起，这表明染色体周围区域具有瞬时性质。还观察到染色体的长程扩散和运动。有趣的是，当细胞用低浓度放线菌素 D 处理时，该药物通过插入富含 GC 的 DNA 抑制 Pol I 转录，染色体运动明显受阻。用另一种不影响 DNA 的 Pol I 抑制剂 metarrestin 处理，对运动的影响很小，这表明 DNA 结构本身在染色体动力学中起作用。此外，用 α-鹅膏蕈碱抑制 Pol II 转录也减少了染色体运动，这表明 Pol II 转录而不是 Pol I 转录对核内染色体动力学很重要。
