Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, California, USA.
Biophys J. 2012 Feb 8;102(3):691-7. doi: 10.1016/j.bpj.2011.11.4026. Epub 2012 Feb 7.
We present a quantitative fluctuation-based assay to measure the degree of local chromatin compaction and investigate how chromatin density regulates the diffusive path adopted by an inert protein in dividing cells. The assay uses CHO-K1 cells coexpressing untagged enhanced green fluorescent protein (EGFP) and histone H2B tagged mCherry. We measure at the single-cell level the EGFP localization and molecular flow patterns characteristic of each stage of chromatin compaction from mitosis through interphase by means of pair-correlation analysis. We find that the naturally occurring changes in chromatin organization impart a regulation on the spatial distribution and temporal dynamics of EGFP within the nucleus. Combined with the analysis of Ca(2+) intracellular homeostasis during cell division, EGFP flow regulation can be interpreted as the result of controlled changes in chromatin compaction. For the first time, to our knowledge, we were able to probe chromatin compaction on the micrometer scale, where the regulation of molecular diffusion may become relevant for many cellular processes.
我们提出了一种基于定量波动的测定方法，以测量局部染色质的紧凑程度，并研究染色质密度如何调节惰性蛋白在分裂细胞中采用的扩散路径。该测定方法使用共表达未标记的增强型绿色荧光蛋白（EGFP）和组蛋白 H2B 标记 mCherry 的 CHO-K1 细胞。我们通过关联分析在单细胞水平上测量染色质紧凑化的各个阶段（从有丝分裂到间期）的 EGFP 定位和分子流动模式。我们发现，染色质组织的自然变化对细胞核内 EGFP 的空间分布和时间动态产生了调节作用。结合细胞分裂期间细胞内 Ca(2+)内稳态的分析，EGFP 流的调节可以解释为染色质紧凑化的控制变化的结果。据我们所知，这是首次在微米尺度上探测染色质的紧凑化，其中分子扩散的调节可能与许多细胞过程相关。
