Zhang C, Jenkins H, Goldberg M W, Allen T D, Hutchison C J
Department of Biological Sciences, University of Dundee, Scotland, UK.
J Cell Sci. 1996 Sep;109 ( Pt 9):2275-86. doi: 10.1242/jcs.109.9.2275.
Nuclear lamina and matrices were prepared from sperm pronuclei assembled in Xenopus egg extracts using a fractionation and extraction procedure. Indirect immunofluorescence revealed that while chromatin was efficiently removed from nuclei during the extraction procedure, the distribution of lamins was unaffected. Consistent with this data, the amount of lamin B3, determined by immunoblotting, was not affected through the extraction procedure. Nuclear matrices were visualised in DGD sections by TEM. Within these sections filaments were observed both at the boundary of the nucleus (the lamina) and within the body of the nucleus (internal nuclear matrix filaments). To improve resolution, nuclear matrices were also prepared as whole mounts and viewed using field emission in lens scanning electron microscopy (FEISEM). This technique revealed two distinct networks of filaments. Filaments lying at the surface of nuclear matrices interconnected nuclear pores. These filaments were readily labelled with monoclonal anti-lamin B3 antibodies. Filaments lying within the body of the nuclear matrix were highly branched but were not readily labelled with antilamin B3 antibodies. Nuclear matrices were also prepared from sperm pronuclei assembled in lamin B3 depleted extracts. Using FEISEM, filaments were also detected in these preparations. However, these filaments were poorly organised and often appeared to aggregate. To confirm these results nuclear matrices were also observed as whole mounts using TEM. Nuclear matrices prepared from control nuclei contained a dense array of interconnected filaments. Many (but not all) of these filaments were labelled with anti-lamin B3 antibodies. In contrast, nuclear matrices prepared from "lamin depleted nuclei' contained poorly organised or aggregated filaments which were not specifically labelled with anti-lamin B3 antibodies.
使用分级分离和提取程序，从非洲爪蟾卵提取物中组装的精子原核制备核纤层和核基质。间接免疫荧光显示，虽然在提取过程中染色质从细胞核中有效去除，但核纤层蛋白的分布未受影响。与该数据一致，通过免疫印迹测定的核纤层蛋白B3的量在提取过程中未受影响。通过透射电子显微镜（TEM）在DGD切片中观察核基质。在这些切片中，在细胞核边界（核纤层）和细胞核内部（内核基质细丝）均观察到细丝。为了提高分辨率，还将核基质制备成整装标本，并使用透镜场发射扫描电子显微镜（FEISEM）进行观察。该技术揭示了两个不同的细丝网络。位于核基质表面的细丝连接核孔。这些细丝很容易用抗核纤层蛋白B3单克隆抗体标记。位于核基质内部的细丝高度分支，但不容易用抗核纤层蛋白B3抗体标记。也从在核纤层蛋白B3缺失提取物中组装的精子原核制备核基质。使用FEISEM，在这些制备物中也检测到细丝。然而，这些细丝组织不良，经常似乎聚集在一起。为了证实这些结果，还使用TEM作为整装标本观察核基质。从对照细胞核制备的核基质包含密集排列的相互连接的细丝。这些细丝中的许多（但不是全部）用抗核纤层蛋白B3抗体标记。相比之下，从“核纤层蛋白缺失的细胞核”制备的核基质包含组织不良或聚集的细丝，这些细丝未用抗核纤层蛋白B3抗体特异性标记。
