XENOPUS LAMIN B-3 HAS A DIRECT ROLE IN THE ASSEMBLY OF A REPLICATION COMPETENT NUCLEUS - EVIDENCE FROM CELL-FREE EGG EXTRACTS

Xenopus egg extracts which assemble replication competent nuclei in vi tro were depleted of lamin B-3 using monoclonal antibody L6 5D5 linked to paramagnetic beads. After depletion, the extracts were still capab le of assembling nuclei around demembranated sperm heads. Using field emission in lens scanning electron microscopy (FEISEM) we show that mo st nuclei assembled in lamin B-3-depleted extracts have continuous nuc lear envelopes and well formed nuclear pores. However, several consist ent differences were observed. Most nuclei were small and only attaine d diameters which were half the size of controls. In a small number of nuclei, nuclear pore baskets, normally present on the inner aspect of the nuclear envelope, appeared on its outer surface. Finally, the ass embly of nuclear pores was slower in lamin B-3-depleted extracts, indi cating a slower overall rate of nuclear envelope assembly. The results of FEISEM were confirmed using conventional TEM thin sections, where again the majority of nuclei assembled in lamin B-3-depleted extracts had well formed double unit membranes containing a high density of nuc lear pores. Since nuclear envelope assembly was mostly normal but slow in these nuclei, the lamin content of 'depleted' extracts was investi gated. While lamin B-3 was recovered efficiently from cytosolic and me mbrane fractions by our procedure, a second minor lamin isoform, which has characteristics similar to those of the somatic lamin B-2, remain ed in the extract. Thus it is likely that this lamin is necessary for nuclear envelope assembly. However, while lamin B-2 did not co-precipi tate with lamin B-3 during immunodepletion experiments, several protei n species did specifically associate with lamin B-3 on paramagnetic im munobeads. The major protein species associated with lamin B-3 migrate d with molecular masses of 102 kDa and 57 kDa, respectively, on one-di mensional polyacrylamide gels. On two-dimensional O'Farrell gels the m obility of the 102 kDa protein was identical to the mobility of a majo r nuclear matrix protein, indicating a specific association between la min B-3 and other nuclear matrix proteins. Nuclei assembled in lamin B -3-depleted extracts did not assemble a lamina, judged by indirect imm unofluorescence, and failed to initiate semi-conservative DNA replicat ion. However, by reinoculating depleted extracts with purified lamin B -3, nuclear lamina assembly and DNA replication could both be rescued. Thus it seems likely that the inability of lamin-depleted extracts to assemble a replication competent nucleus is a direct consequence of a failure to assemble a lamina.