Martelli A M, Falcieri E, Gobbi P, Manzoli L, Gilmour R S, Cocco L
Istituto di Anatomia Umana Normale, Bologna, Italy.
Exp Cell Res. 1991 Oct;196(2):216-25. doi: 10.1016/0014-4827(91)90254-r.
Using mouse erythroleukemia cells we performed a comprehensive morphological and biochemical study of the nuclear matrix obtained after exposure of isolated nuclei to 37 degrees C or from cells heat shocked in vivo at 43 or 45 degrees C. At the ultrastructural level it was possible to see that in the absence of a 37 degrees C incubation of purified nuclei, the final matrix lacked well-defined nucleolar remnants but a peripheral lamina was clearly visible, as well as a sparse fibrogranular network which was located at the periphery of the structures. On the contrary, after a 37 degrees C nuclear incubation, very electron-dense nucleolar remnants were observed along with an abundant meshwork dispersed throughout the interior of the structures. When intact cells were heat shocked in vivo, electron-dense residual nucleoli were present only when isolated nuclei had been exposed to 37 degrees C in vitro, whereas without such an incubation, they were not as easily distinguishable and appeared less electron-dense. In the latter case the inner network was more evenly distributed. After purified nuclei were incubated at 37 degrees C for 45 min, the high salt and DNase I resistant fraction retained about 18% of the nuclear protein whereas if the heating was omitted protein recovery dropped to 6%. An increase in the recovery of intact structures in the matrix fraction was the main reason for the higher protein recovery. Heating nuclei in vitro further increased the amount of nuclear protein present in the matrix fraction even if intact cells had been heat shocked in vivo. No major qualitative differences were seen when the polypeptide pattern of the various types of nuclear matrices was analyzed on one-dimensional polyacrylamide gels and this finding was further supported by Western blot analysis with a monoclonal antibody to lamins A and C. These results show that heating mainly stabilizes the nucleolar remnants of the matrix and to a lesser extent the inner network, but the morphology of the final structures is different depending on whether the stabilization is performed in vivo or in vitro.
我们使用小鼠红白血病细胞，对分离的细胞核在37℃下孵育后或体内在43℃或45℃热休克处理的细胞所获得的核基质进行了全面的形态学和生化研究。在超微结构水平上可以看到，纯化的细胞核在没有37℃孵育的情况下，最终的核基质缺乏明确的核仁残余物，但可见外周核纤层，以及位于结构周边的稀疏纤维颗粒网络。相反，在37℃进行细胞核孵育后，观察到非常电子致密的核仁残余物以及遍布结构内部的丰富网络。当完整细胞在体内进行热休克处理时，只有在体外将分离的细胞核暴露于37℃时才会出现电子致密的残余核仁，而没有这种孵育时，它们不太容易区分且电子密度较低。在后一种情况下，内部网络分布更均匀。纯化的细胞核在37℃孵育45分钟后，高盐和DNase I抗性组分保留了约18%的核蛋白，而如果省略加热，蛋白质回收率降至6%。基质组分中完整结构回收率的增加是蛋白质回收率较高的主要原因。即使完整细胞在体内已经进行过热休克处理，但体外加热细胞核进一步增加了基质组分中存在的核蛋白量。当在一维聚丙烯酰胺凝胶上分析各种类型核基质的多肽模式时，未观察到主要质量差异，并且用抗核纤层蛋白A和C的单克隆抗体进行的蛋白质印迹分析进一步支持了这一发现。这些结果表明，加热主要稳定核基质的核仁残余物，对内部网络的稳定作用较小，但最终结构的形态取决于稳定作用是在体内还是体外进行。
