Maniotis A J, Bojanowski K, Ingber D E
Department of Pathology, Children's Hospital, Boston, MA 02115, USA.
J Cell Biochem. 1997 Apr;65(1):114-30.
Chromatin is thought to be structurally discontinuous because it is packaged into morphologically distinct chromosomes that appear physically isolated from one another in metaphase preparations used for cytogenetic studies. However, analysis of chromosome positioning and movement suggest that different chromosomes often behave as if they were physically connected in interphase as well as mitosis. To address this paradox directly, we used a microsurgical technique to physically remove nucleoplasm or chromosomes from living cells under isotonic conditions. Using this approach, we found that pulling a single nucleolus or chromosome out from interphase or mitotic cells resulted in sequential removal of the remaining nucleoli and chromosomes, interconnected by a continuous elastic thread. Enzymatic treatments of interphase nucleoplasm and chromosome chains held under tension revealed that mechanical continuity within the chromatin was mediated by elements sensitive to DNase or micrococcal nuclease, but not RNases, formamide at high temperature, or proteases. In contrast, mechanical coupling between mitotic chromosomes and the surrounding cytoplasm appeared to be mediated by gelsolin-sensitive microfilaments. Furthermore, when ion concentrations were raised and lowered, both the chromosomes and the interconnecting strands underwent multiple rounds of decondensation and recondensation. As a result of these dynamic structural alterations, the mitotic chains also became sensitive to disruption by restriction enzymes. Ion-induced chromosome decondensation could be blocked by treatment with DNA binding dyes, agents that reduce protein disulfide linkages within nuclear matrix, or an antibody directed against histones. Fully decondensed chromatin strands also could be induced to recondense into chromosomes with pre-existing size, shape, number, and position by adding anti-histone antibodies. Conversely, removal of histones by proteolysis or heparin treatment produced chromosome decondensation which could be reversed by addition of histone H1, but not histones H2b or H3. These data suggest that DNA, its associated protein scaffolds, and surrounding cytoskeletal networks function as a structurally-unified system. Mechanical coupling within the nucleoplasm may coordinate dynamic alterations in chromatin structure, guide chromosome movement, and ensure fidelity of mitosis.
染色质被认为在结构上是不连续的，因为它被包装成形态上不同的染色体，在用于细胞遗传学研究的中期制备物中，这些染色体在物理上似乎彼此隔离。然而，对染色体定位和运动的分析表明，不同的染色体在间期以及有丝分裂期的行为常常就好像它们在物理上是相连的。为了直接解决这一矛盾，我们使用了一种显微手术技术，在等渗条件下从活细胞中物理去除核质或染色体。通过这种方法，我们发现从间期或有丝分裂细胞中拉出单个核仁或染色体，会导致其余核仁和染色体依次被去除，它们由一条连续的弹性细丝相连。对处于张力下的间期核质和染色体链进行酶处理后发现，染色质内的机械连续性是由对DNA酶或微球菌核酸酶敏感的成分介导的，但对RNA酶、高温下的甲酰胺或蛋白酶不敏感。相反，有丝分裂染色体与周围细胞质之间的机械偶联似乎是由凝溶胶蛋白敏感的微丝介导的。此外，当离子浓度升高和降低时，染色体和连接链都会经历多轮解聚和再凝聚。由于这些动态结构变化，有丝分裂链也变得对限制酶的破坏敏感。离子诱导的染色体解聚可以通过用DNA结合染料、减少核基质内蛋白质二硫键的试剂或针对组蛋白的抗体处理来阻断。通过添加抗组蛋白抗体，完全解聚的染色质链也可以被诱导重新凝聚成具有预先存在的大小、形状、数量和位置的染色体。相反，通过蛋白水解或肝素处理去除组蛋白会导致染色体解聚，添加组蛋白H1可以使其逆转，但添加组蛋白H2b或H3则不能。这些数据表明，DNA、其相关的蛋白质支架和周围的细胞骨架网络作为一个结构统一的系统发挥作用。核质内的机械偶联可能协调染色质结构的动态变化，引导染色体运动，并确保有丝分裂的准确性。
