Goldberg M W, Blow J J, Allen T D
CRC Department of Structural Cell Biology, Paterson Institute for Cancer Research, Christie Hospital, Holt Radium Institute, Manchester, United Kingdom.
J Struct Biol. 1992 May-Jun;108(3):257-68. doi: 10.1016/1047-8477(92)90026-7.
At mitosis the nuclear envelope (NE) is disassembled to allow chromosome separation. In telophase it is reassembled as the chromosomes decondense. Cell-free extracts of Xenopus eggs have been used extensively to study assembly of the NE and the nuclear pore complexes (NPCs), providing several models for the steps involved. The NE is a surface structure which in cell-free extracts is easily exposed. It is appropriate, therefore, to use a surface imaging technique to study NE dynamics. Field emission in-lens scanning electron microscopy (FEISEM) provides the opportunity to image surfaces, directly, and to visualise details of structures such as the NPC. Here we show the feasibility and value of FEISEM to study the steps of NE formation. Nuclei have been assembled in vitro and fixed at different time points during assembly, followed by conductive staining, platinum coating, and visualisation by FEISEM. Changes on the nuclear surface with time are shown. Details of the surface of chromatin and the cytoplasmic face of NPC structure are demonstrated without the need to isolate the structures from the nucleus.
在有丝分裂时，核膜（NE）解体以允许染色体分离。在末期，随着染色体解聚，核膜重新组装。非洲爪蟾卵的无细胞提取物已被广泛用于研究核膜和核孔复合体（NPC）的组装，为其中涉及的步骤提供了几种模型。核膜是一种表面结构，在无细胞提取物中很容易暴露出来。因此，使用表面成像技术来研究核膜动态是合适的。场发射透镜内扫描电子显微镜（FEISEM）提供了直接对表面成像并可视化诸如核孔复合体等结构细节的机会。在这里，我们展示了FEISEM研究核膜形成步骤的可行性和价值。已在体外组装细胞核，并在组装过程中的不同时间点进行固定，随后进行导电染色、铂涂层处理，并通过FEISEM进行可视化。展示了核表面随时间的变化。无需从细胞核中分离结构，就可以展示染色质表面和核孔复合体结构胞质面的细节。
