Department of Mechanical Engineering, The University of Tokyo, Tokyo, 113-8656, Japan.
Institute for Frontier Life & Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan.
Sci Rep. 2018 Sep 12;8(1):13684. doi: 10.1038/s41598-018-31975-5.
Chromatin folding shows spatio-temporal fluctuations in living undifferentiated cells, but fixed spatial heterogeneity in differentiated cells. However, little is known about variation in folding stability along the chromatin fibres during differentiation. In addition, effective methods to investigate folding stability at the single cell level are lacking. In the present study, we developed a microfluidic device that enables non-destructive isolation of chromosomes from single mammalian cells as well as real-time microscopic monitoring of the partial unfolding and stretching of individual chromosomes with increasing salt concentrations under a gentle flow. Using this device, we compared the folding stability of chromosomes between non-differentiated and differentiated cells and found that the salt concentration which induces the chromosome unfolding was lower (≤500 mM NaCl) for chromosomes derived from undifferentiated cells, suggesting that the chromatin folding stability of these cells is lower than that of differentiated cells. In addition, individual unfolded chromosomes, i.e., chromatin fibres, were stretched to 150-800 µm non-destructively under 750 mM NaCl and showed distributions of highly/less folded regions along the fibres. Thus, our technique can provide insights into the aspects of chromatin folding that influence the epigenetic control of cell differentiation.
染色质折叠在未分化的活细胞中表现出时空波动，但在分化的细胞中呈现固定的空间异质性。然而，关于分化过程中沿着染色质纤维折叠稳定性的变化知之甚少。此外，缺乏在单细胞水平上研究折叠稳定性的有效方法。在本研究中，我们开发了一种微流控装置，该装置能够从单个哺乳动物细胞中非破坏性地分离染色体，并在温和的流动下实时监测随着盐浓度增加，单个染色体的部分展开和拉伸。使用该装置，我们比较了未分化和分化细胞中染色体的折叠稳定性，发现诱导染色体展开的盐浓度较低（≤500mM NaCl），来自未分化细胞的染色体，表明这些细胞的染色质折叠稳定性低于分化细胞。此外，在 750mM NaCl 下，单个展开的染色体（即染色质纤维）可无损地拉伸至 150-800µm，并显示出纤维上高度/较少折叠区域的分布。因此，我们的技术可以深入了解影响细胞分化的表观遗传控制的染色质折叠方面。
