Density imaging of heterochromatin in live cells using orientation-independent-DIC microscopy.
Density imaging of heterochromatin in live cells using orientation-independent-DIC microscopy.
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DOI：
10.1091/mbc.e17-06-0359
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发表时间：
2017-11-07
影响因子：
3.3
通讯作者：
Maeshima K
中科院分区：
文献类型：
--
作者：
Imai R;Nozaki T;Tani T;Kaizu K;Hibino K;Ide S;Tamura S;Takahashi K;Shribak M;Maeshima K
使用方向无关DIC显微镜，我们发现，异染色质中的总材料的密度仅为1.53倍高于常染色质，而DNA密度为7.5倍。这种令人惊讶的小差异可能是由于两种染色质中蛋白质和RNA的优势，这可能有助于对异染色质产生适度的屏障。在真核细胞中，高度浓缩的失活/沉默的染色质长期以来被称为“异染色质”。然而，最近的研究表明，这些区域实际上并不是完全转录沉默的，并且对异染色质只存在适度的进入障碍。为了进一步研究这个问题，关键是要阐明异染色质的物理性质，如其在活细胞中的总密度。在这里，使用方向无关的微分干涉对比（OI-DIC）显微镜，这是能够映射光路差异，我们调查了密度的总材料在pericentric焦点，一个代表性的异染色质模型，在活小鼠NIH 3 T3细胞。我们证明，异染色质的总密度（208毫克/毫升）仅为1.53倍高于周围的常染色质区域（136毫克/毫升），而异染色质的DNA密度是5.5至7.5倍高。我们观察到在典型的兼性异染色质（失活的人类X染色体）中类似的微小密度差异。这种令人惊讶的小差异可能是由于非核小体物质（蛋白质/RNA）（约120 mg/ml）在两个染色质区域中占主导地位。蒙特卡罗模拟表明，非核小体材料有助于创造一个适度的访问障碍异染色质，允许最小的蛋白质进入功能区。我们的OI-DIC成像为活细胞环境提供了新的见解。
Using orientation-independent-DIC microscopy, we revealed that the density of total materials in heterochromatin was only 1.53-fold higher than that of euchromatin, whereas the DNA density was 7.5-fold higher. This surprisingly small difference may be due to the dominance of proteins and RNAs in both chromatins, which may help create a moderate barrier to heterochromatin. In eukaryotic cells, highly condensed inactive/silenced chromatin has long been called “heterochromatin.” However, recent research suggests that such regions are in fact not fully transcriptionally silent and that there exists only a moderate access barrier to heterochromatin. To further investigate this issue, it is critical to elucidate the physical properties of heterochromatin such as its total density in live cells. Here, using orientation-independent differential interference contrast (OI-DIC) microscopy, which is capable of mapping optical path differences, we investigated the density of the total materials in pericentric foci, a representative heterochromatin model, in live mouse NIH3T3 cells. We demonstrated that the total density of heterochromatin (208 mg/ml) was only 1.53-fold higher than that of the surrounding euchromatic regions (136 mg/ml) while the DNA density of heterochromatin was 5.5- to 7.5-fold higher. We observed similar minor differences in density in typical facultative heterochromatin, the inactive human X chromosomes. This surprisingly small difference may be due to that nonnucleosomal materials (proteins/RNAs) (∼120 mg/ml) are dominant in both chromatin regions. Monte Carlo simulation suggested that nonnucleosomal materials contribute to creating a moderate access barrier to heterochromatin, allowing minimal protein access to functional regions. Our OI-DIC imaging offers new insight into the live cellular environments.