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Fig. 1. (A) Comparison of soluble
proteins, biotinylated surface
proteins and proteins recognized
on western blot by antibodies 181
in Xenopus stage 6 oocytes. Lane
a, soluble protein extracts prepared
from oocytes separated in
a polyacrylamide gel and stained
with Coomassie blue. Lane b,
oocyte surface proteins were
labeled with NH8-biotin as described
in Materials and Methods.
The extracts were electrophoresed
and blotted and the
blots were reacted with the
streptavidin-horseradish peroxidase
conjugate. A maximum of
14 polypeptides with apparent Mr
kDa
kDa 94_
67_
_ 76
ii -63
43_
'il!\@ll 30_ li _38
20_
_28 14_
a b c
--
~
a b
kDal
94 67_ <·- 43-,,:. · - 30_
20_ • a c
I
• d
-56
... 40 -as
-32
ranging from 20 to 115 kDa react to the conjugate. Lane c, oocyte soluble proteins separated in a polyacrylamide gel, transferred on
nitrocellulose sheet and labeled with antibodies IS1 and with secondary peroxidase coupled antibodies. 181 reacts with 10 antigenic
components with apparent Mr ranging from 20 to 76 kDa. As shown here, only polypeptides with apparent Mr ranging from about 38
to 60 kDa react strongly. Note that 181 exclusively recognizes plasma membrane proteins. Compare with lane b. (B) 808-PAGE of
biotin-linked purified actin (lane a) and proteins extracted from biotinylated Xenopus oocytes (lane b). Actin bands do not correspond
to biotin-linked polypeptides of oocytes (arrows), indicating that only surface proteins were bound to biotin during incubation of oocytes
in the presence of the labeling agent. (C) Comparison of proteins recognized on immunoblots by antibodies IS1 in Xenopus unfertilized
eggs and erythrocytes. Lanes a and b, plasma membranes from eggs and erythrocytes; lanes c and d, vitelline membrane proteins
and yolk. The antibodies 181 recognize various proteins of the egg and erythrocyte plasma membranes and of the vitelline
membrane but none of the yolk. The arrows and arrowheads point to the positions of the major proteins in the extracts and their molecular
masses, respectively. In A-C, molecular mass markers (in kDa) are shown at the left of the lanes.
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Fig 2. lmmunostaining of sections of Xenopus ovary, containing
oocytes at different stages after reaction with antibodies IS1.
Upper part, the plasma membranes of the previtellogenic and
vitellogenic oocytes are labeled (arrowheads). The germinal
vesicles (arrow) show no reaction with the antibodies. Bar 50 IJm.
Lower part: specific intense staining of the plasma membrane (arrowhead)
of a mature oocyte. The light staining of yolk is caused
by its autofluorescence. Bar, 151-Jm.
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Fig. 3. lmmunostaining of Xenopus
egg meridian section (animal
hemisphere to the top) with antibodies
IS1 at different phases of
the first division cycle (immunofluorescence,
except D and E,
peroxidase staining on albino
eggs). (A) General view of the animal
pole of an egg showing the
labeling of the plasma membrane
(arrow) by the antibodies IS1. The
staining of the inner hyalin cytoplasm
and yolk platelets is because
of their autofluorescence.
(B) Section showing the label of
the plasma membrane (large
arrow) and a slight staining, appearing
as free dots, in the inner
cytoplasm (small arrows) at NT
0.60. (C.D) Accumulation of cytoplasmic
stained materials (arrow)
under the plasma membrane
(large arrow in C) at NT 0.80. In
D, the fertilization layer is also
stained (small arrow). (E,F)
Sections of eggs at the time of the
first cleavage (NT 1 0). Staining is
essentially located underneath the
furrow edge of the nascent furrow
(arrow in E). As the furrow deepens,
labeled vesicles accumulate
just above the furrow base in the
presumed zone of plasma membrane
insertion (ZPMA; arrows in
F). The new plasma membrane of
the furrow walls is also stained (E).
(G) Section of a fertilized egg incubated
in the presence of cycloheximide
and fixed at NT 1.05.
The inhibitor causes a drastic
depletion of plasma membrane
precursors. The labeled material
is widely dispersed as aggregates throughout the inner cytoplasm (arrows); compare with E. (H) Section of a fertilized egg incubated
in the presence of colchicine and fixed at 145 min PF. The stained material is collapsed and forms radial aggregates into the cytoplasm
(arrows). (I) Activated egg fixed at NT 1.0 showing an intense and heterogenous distribution of the label in areas of the cortex
where no furrow develops (arrows). Presumably because of the absence of a coherent microtubular system, the labeled materials cannot
be transported towards the furrow and remain dispersed under the plasma membrane. Bars, A: 40 um; B, H: 25 um; C,F-1: 15 um;
D,E: 150um.
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Fig. 4. Electron microscopic
immunogold localization ( 10 nm
gold particles) on sections
through a first furrow of Xenopus
egg alter reaction with antibodies
IS1. (A) General view of one side
of a nascent furrow (animal pole
at the top of the figure). Upper
part, surface of the egg; middle
part (between arrows), furrow wall
with crypts and convoluted microvilli
(MV); lower part, bottom of
the furrow displaying a smooth
plasma membrane. Because of
the low magnification, staining
(10 nm particles) is not visible.
(B-D) Micrographs showing the
different zones of a nascent furrow,
15 )Jm in depth, at a higher
magnification. (B,C) Furrow wall.
In (B) note the association of
immunogold particles with the MV
and crypts (arrow) of the plasma
membrane of the furrow wall and
with intracytoplasmic vesicles
(arrowheads). Note in (C) the
close association of a stained
cytoplasmic vesicle with the
plasma membrane (arrow). (D)
Furrow base. Labeling of the
plasma membrane of the furrow
base. Note the absence of
labeled intracytoplasmic vesicles.
The light labeling is the result
of using IS1 antibodies preabsorbed
with Xenopus erythrocytes (see Materials and Methods). Compare with (C), illustrating the antigenic community between
proteins of egg and erythrocyte surfaces. (E) Egg surface. One hundred micrometers outside the furrow, a small vesicle (arrow), stained
with IS1, is present under the labeled plasma membrane. Labeling the same as for (D). (F) General view of a furrow, 35 )Jm in depth.
The external edges and the central zone of the furrow consist of smooth plasma membrane. Just above the leading edge of the furrow,
the plasma membrane of the walls is folded because of the presence of MV (arrows). This corresponds, as in (A), to the zone referred
to as the zone of plasma membrane addition (ZPMA) Sections of MV (circular structures) are present in the cleavage furrow. Bars,
A: 1.5 )um; B,C: 0.5 )um, D,E: 1 )um: F: 5 )um.
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Fig. 5. Diagram showing the postulated process of plasma membrane
insertion into the furrow walls during the first egg division
and the role of the microtubule arrays. Pre-cleavage phase: (A)
Recruitment and then accumulation of plasma membrane precursors
(intracytoplasmic vesicles) at the animal pole of the egg. (B,C)
The two sets of microtubule arrays carry the membrane precursors
to the predetermined furrow formation site. Cleavage phase:
(D) Nascent furrow. Insertion of the intracytoplasmic vesicles onto
the edges of the furrow at the zone of plasma membrane addition
(ZPMA), guidance of the precursors by the microtubule arrays (E)
Deepening of the furrow groove in the animal hemisphere. The
ZPMA is near the base of the furrow. (E,F) Completion of egg
division with extension of the furrow in the vegetative hemisphere.
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