J Cell Biol
November 1, 1993;
Membrane-associated lamins in Xenopus egg extracts: identification of two vesicle populations.
Nuclear lamin isoforms of vertebrates can be divided into two major classes. The B-type lamins are membrane associated throughout the cell cycle, whereas A-type lamins are recovered from mitotic cell
homogenates in membrane-free fractions. A feature of oogenesis in birds and mammals is the nearly exclusive presence of B-type lamins in oocyte
nuclear envelopes. In contrast, oocytes and early cleavage
embryos of the amphibian Xenopus laevis are believed to contain a single lamin isoform, lamin LIII
, which after nuclear envelope breakdown during meiotic maturation is reported to be completely soluble. Consequently, we have reexamined the lamin complement of Xenopus oocyte
nuclear envelopes, egg
extracts, and early embryos. An mAb (X223) specific for the homologous B-type lamins B2 of mouse and LII of Xenopus somatic cells (Höger, T., K. Zatloukal, I. Waizenegger, and G. Krohne. 1990. Chromosoma. 99:379-390) recognized a Xenopus oocyte
nuclear envelope protein biochemically distinct from lamin LIII
and very similar or identical to somatic cell lamin LII. Oocyte
lamin LII was detectable in nuclear envelopes of early cleavage
embryos. Immunoblotting of fractionated egg
extracts revealed that approximately 20-23% of lamin LII and 5-7% of lamin LIII
were membrane associated. EM immunolocalization demonstrated that membrane-bound lamins LII and LIII are associated with separate vesicle populations. These findings are relevant to the interpretation of nuclear reconstitution experiments using Xenopus egg
J Cell Biol
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References [+] :
Figure I. Identification of Xenopus lamin Ln in oocyte nuclear
envelopes. Nuclear envelopes from 50 oocytes (A-D), or follicle
ceils derived from 25 oocytes (E) were salt washed and proteins
electrophoretically separated by two-dimensional gel electrophoresis
(first dimension, NEPHGE; second dimension, SDS-PAGE).
Proteins were transferred to nitrocellulose filters and stained with
Ponceau S to determine the position of the coelectrophoresed
marker proteins actin, BSA, and PGK. The filters were probed with
the antilamin Lm antibody 46F7 (A), the antilamin LII antibody
X223 (B and E), or the antilamin Ln/Lm antibody X155 (D). The
filter shown in A was reprobed with the antilamin LII antibody
X223 (C). Arrowheads (A-C and F) mark the position of the quantitatively
major isoelectric variant of oocyte lamin Ln. Brackets
(D), indicate the isoelectric variants of L~ and Lm recognized by
the antibody X155.
Figure 2. Immunolocalization of lamin La in ovary. Indirect immunofluorescence microscopy on Xenopus ovary sections after incubation
with antilamin Lm antibody 46F7 (A) or antilamin Lu antibody X223 (B). Chromatin staining (A~ B') by HOECHST. Bar, 50um.
Figure 3. EM immunolocalization of lamin L~ to the oocyte nuclear
lamina. Isolated oocyte nuclear envelopes were incubated
with antilamin LI~ antibody X223 followed by secondary antibodies
conjugated to 10-nm colloidal gold. Gold particles are observed
exclusively on the nucleoplasmic (N) side of the envelope. Nuclear
pore complexes are marked by arrowheads. C, cytoplasmic side.
Bar, 0.2 um.
Figure 4. Immunolocalization of lamins in two-cell-stage embryos by indirect immunofluorescence microscopy. Squashed preparations
of in vitro fertilized embryos halted in interphase at the two-cell stage by cycloheximide were incubated with antilamin Lm antibody 46F7
(A) or antilamin LI~ antibody X223 (B), followed by Texas red-conjugated secondary antibodies. Chromatin staining (.4' and B') by
HOECHST. Bar, 50um.
Figure 5. EM analysis of egg extract fractionated by ultracentrifugation.
Representative examples of materials found in the S~oo Sup.
(A), $2oo Cytosol (B), and $2oo Pellet (C) are shown. Bar, 0.2 um.
Figure 6. Distribution of nuclear envelope proteins in egg extract
fractionated by ultracentrifugation. Proteins from the $1o0 Sup.
(lane 1 ), serial dilutions of $2oo Cytosol (lanes 2-5), and $20o Pellet
(lanes 6-8) were separated by 11% SDS-PAGE. Proteins were
transferred to nitrocellulose filters then probed with anti-gp62 antiserum
(A), anti-p54 antiserum (B), antilamin L./L,I antibody
X155 (C), and antilamin Lm antibody 46F7 (D). Units refers to
the relative amount of St0o Sup. (1.0 U equals 2.4 #1 of Si0o Sup.,
lane 1) contained in the $20o Cytosol (lanes 2-5) and $20o Pellet
(lanes 6-8) aliquots used for gel separation.
Figure 7. Immunoabsorption of vesicles to magnetic beads coated
with antilamin antibodies. EM analysis of vesicles from S~0o Sup.
immunoabsorbed to magnetic beads coated with control goat
anti-mouse antibody (A), antilamin L~I antibody X223 (B), or antilamin
Lm antibody 46F7 (C; D, higher magnification image of
46F7-absorbed vesicles). Bars, 0.2 #m (A-C); 0.1 #m (D).
Figure 8. Immunological
analysis of lamins absorbed
to antibody-coated magnetic
beads. An aliquot of S~0o
Sup.- (lane 1 ) and proteins absorbed
to control (lane 2)-, antilamin
Lm antibody 46F7
(lane 3)-, and antilamin Ln
antibody X223 (lane 4)-
coated magnetic beads were
solubilized, separated by 8 %
SDS-PAGE, then transferred
to nitrocellulose filters for
probing with the antilamin
LII/Lm antibody X155. Arrowheads
indicate the migration
position of the antibody
heavy chains (HC) and lamins
Lm and Ln are noted on the
right of the panel.
Figure 9. EM immunolocalization of lamins Ln and Lm on membranes present in the $200 Pellet. Membranes were immobilized onto glass
coverslips and incubated with the antibodies X223 (A-E) or 46F7 (F-L). Bound antibodies were visualized with secondary antibodies conjugated
to 12-nm colloidal gold. Overviews A (X223) and F (46F7) are shown at identical magnifications. Arrows in the overviews (A
and F) designate vesicles labeled with gold particles. Higher magnification images of antibody X223-1abeled vesicles (B-E) and 46FTlabeled
vesicles (G-L) are also presented. Bars, 0.2 #m (A and F); 0.1 #m (B-E and G--L).
Bailer, Characterization of A 54-kD protein of the inner nuclear membrane: evidence for cell cycle-dependent interaction with the nuclear lamina. 1991, Pubmed