Chaly N, Little J E, Brown D L
Can J Biochem Cell Biol. 1985 Jun;63(6):644-53. doi: 10.1139/o85-082.
Nuclear matrix structure closely resembles the organization of nonchromatin components of nuclei in situ. However, reports on the extent to which nuclear components are reorganized during matrix isolation have produced conflicting results, and the reality of an in situ nuclear matrix is still in question. We have prepared nuclear matrices by processing cells still attached to the growth substrate through the extraction steps, thus avoiding mechanical disruption due to homogenization and centrifugation. Furthermore, the extensive residual cytoskeleton seems to keep the residual nuclei "stretched out" so that they retain many features of intact nuclei. Indirect immunofluorescence staining was used to compare the distribution of nuclear antigens in intact nuclei with their organization in nuclear matrices, as well as at each stage of nuclear matrix preparation. We have applied monoclonal antibodies P1, I1, PI1, and PI2, which had been generated against isolated matrices, as well as autoimmune sera detecting lamins, perichromin, and centromere antigens. Chromatin and RNA extraction was monitored with Hoechst 33258, ethidium bromide, and antihistone. The lamins, PI1, and, to a great extent, PI2 and centromere antigens were little affected by the extraction. The data suggest furthermore that PI1 is a fundamental nuclear matrix component and may serve in integrating peripheral and internal nuclear functions. P1 and perichromin were extensively redistributed after chromatin extraction, supporting a role for these antigens in spatial ordering of chromatin. I1 was progressively extracted at each stage of nuclear matrix preparation and was artifactually associated with matrices which had not been digested with RNase. This study demonstrates unequivocally that the organization of many nuclear matrix components in final preparations reflects their organization in situ. It does indicate, however, that some components retained in matrices are extensively redistributed during nuclear matrix preparation and that their role in nuclear organization must be evaluated in consequence.
核基质结构与细胞核中非染色质成分在原位的组织形式极为相似。然而，关于在核基质分离过程中核成分的重组程度的报道却产生了相互矛盾的结果，并且原位核基质的真实性仍存在疑问。我们通过对仍附着在生长底物上的细胞进行提取步骤来制备核基质，从而避免了因匀浆和离心导致的机械破坏。此外，广泛存在的残余细胞骨架似乎使残余细胞核“伸展”，从而使其保留了完整细胞核的许多特征。使用间接免疫荧光染色来比较完整细胞核中核抗原的分布与其在核基质以及核基质制备的每个阶段中的组织形式。我们应用了针对分离的基质产生的单克隆抗体P1、I1、PI1和PI2，以及检测核纤层蛋白、染色质周蛋白和着丝粒抗原的自身免疫血清。用Hoechst 33258、溴化乙锭和抗组蛋白监测染色质和RNA的提取。核纤层蛋白、PI1，以及在很大程度上PI2和着丝粒抗原受提取的影响较小。数据进一步表明，PI1是一种基本的核基质成分，可能在整合细胞核外周和内部功能中发挥作用。染色质提取后，P1和染色质周蛋白广泛重新分布，支持了这些抗原在染色质空间排列中的作用。在核基质制备的每个阶段，I1都逐渐被提取，并与未用核糖核酸酶消化的基质产生人为关联。这项研究明确表明，最终制备物中许多核基质成分的组织形式反映了它们在原位的组织形式。然而，这确实表明，保留在基质中的一些成分在核基质制备过程中会广泛重新分布，因此必须相应地评估它们在核组织中的作用。
