School of Physics and Astronomy, University of Minnesota, Minneapolis MN, USA.
Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester MN, USA.
Nucleic Acids Res. 2022 Jul 22;50(13):e78. doi: 10.1093/nar/gkac314.
The dynamic rearrangement of chromatin is critical for gene regulation, but mapping both the spatial organization of chromatin and its dynamics remains a challenge. Many structural conformations are too small to be resolved via conventional fluorescence microscopy and the long acquisition time of super-resolution photoactivated localization microscopy (PALM) precludes the structural characterization of chromatin below the optical diffraction limit in living cells due to chromatin motion. Here we develop a correlative conventional fluorescence and PALM imaging approach to quantitatively map time-averaged chromatin structure and dynamics below the optical diffraction limit in living cells. By assigning localizations to a locus as it moves, we reliably discriminate between bound and unbound dCas9 molecules, whose mobilities overlap. Our approach accounts for changes in DNA mobility and relates local chromatin motion to larger scale domain movement. In our experimental system, we show that compacted telomeres move faster and have a higher density of bound dCas9 molecules, but the relative motion of those molecules is more restricted than in less compacted telomeres. Correlative conventional and PALM imaging therefore improves the ability to analyze the mobility and time-averaged nanoscopic structural features of locus specific chromatin with single molecule sensitivity and yields unprecedented insights across length and time scales.
染色质的动态重排对于基因调控至关重要，但绘制染色质的空间组织及其动态仍然是一个挑战。许多结构构象太小，无法通过传统的荧光显微镜来解析，而超分辨率光激活定位显微镜（PALM）的长获取时间由于染色质运动而排除了活细胞中低于光衍射极限的染色质的结构特征。在这里，我们开发了一种相关的常规荧光和 PALM 成像方法，以定量绘制活细胞中低于光衍射极限的染色质的时间平均结构和动态。通过在其运动时将局部定位分配给一个基因座，我们可以可靠地区分结合和未结合的 dCas9 分子，它们的流动性重叠。我们的方法考虑了 DNA 流动性的变化，并将局部染色质运动与更大规模的域运动联系起来。在我们的实验系统中，我们表明，压缩的端粒移动得更快，并且具有更高密度的结合 dCas9 分子，但这些分子的相对运动比不太压缩的端粒受到更多限制。因此，相关的常规和 PALM 成像提高了以单分子灵敏度分析特定基因座染色质的流动性和时间平均纳米结构特征的能力，并在长度和时间尺度上产生了前所未有的见解。
