Nuclear Envelope and Nuclear Pore Complex Dynamjcs in vitro, Visualised by FEISEM

Extract

As the defining structure in eukaryote cells, the nuclear envelope is completely dismantled and reformed within an hour or so at each cell division (open mitosis). In yeast and some insect tissues, closed mitosis occurs, in which the nuclear envelope is maintained largely intact throughout chromosome separation. Use of cell free systems has allows us access to the mechanisms of cell division and NE dynamics in vitro by FEISEM (Field Emission In Lens Scanning Electron Microscopy. We have used demembranated Xenopus sperm heads as a source of DNA, which is incubated in an extract of Xenopus egg cytoplasm, where it becomes assembled into a normal nucleus with functional nuclear envelope (1-4). DNA replication proceeds under normal cell cycle controls, followed by an in vitro mitosis in suitable conditions. The cytoplasmic extract can be separated into membrane and soluble fractions that can be supplemented with, or depleted of, specific proteins. Inhibitors and other effectors can be .added to modulate both assembly and transport (5). Using the lectin WGA we have depleted Xenopus cytoplasmic extract of the major nucleoporins, CAN, Nup 98 and p62 and their associated proteins, whose removal effectively inhibits three aspects of nuclear formation, namely NPC formation, nuclear growth, and the reorganisation of the DNA in the depleted nuclei. Adding back these eluted nucleoporins restores normality with respect to nuclear growth, DNA reorganisation and NPC assembly. Current work involves purification of complexes containing these proteins by HPLC to allow add back of the complexes singly and in combination, to characterise their individual roles in NPC assembly, structure and transport (Figures 1,2,3).