M. Goldberg et al., XENOPUS LAMIN B-3 HAS A DIRECT ROLE IN THE ASSEMBLY OF A REPLICATION COMPETENT NUCLEUS - EVIDENCE FROM CELL-FREE EGG EXTRACTS, Journal of Cell Science, 108, 1995, pp. 3451-3461
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.