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Fig. 1. Velocity of yolk platelets throughout cortical rotation, in a
plane perpendicular to the animal-vegetal axis and 8 mm inside the
plasma membrane, at the vegetal pole of a fertilized, immobilized
egg. Four phases of cortical rotation can be distinguished: (1)
initiation - onset of slow movements between 0.28 and 0.40 NT; (2)
acceleration - increase of velocity between 0.40 and 0.50 NT; (3)
translocation - continuation of maximal velocity between 0.50 and
0.85 NT, as the cortex undergoes the bulk of its displacement relative
to the inner cytoplasmic core; and (4) termination - rapid
deceleration after 0.85 NT, sometimes followed by slight reversal of
direction.
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Fig. 2. Single optical sections 8 mm inside the
plasma membrane at the vegetal pole of a
fertilized, immobilized egg, showing the initial
slow displacement of Nile Red-labeled yolk
platelets between (A) 0.27 NT and (B) 0.45 NT.
Yolk platelets have traveled an average of 18
mm in the 14.3 minute time interval between the
images; the black arrow indicates the earlier
position and the white arrow the later position,
of one representative yolk platelet. Bar, 12 mm.
1284
we began our measurements where yolk platelet movements
could first be detected (approximately 4-5 mm inside the
plasma membrane, as shown in the optical section perpendicular
to the coverslip in Fig. 5), then we continued to take
measurements at increasing depths until fluorescent platelets
could no longer be detected (approximately 14-15 mm inside
the plasma membrane).
In one egg for which we obtained extensive data, we found
that the average velocity of small to medium-sized yolk
platelets (£5 mm diameter) in the most peripheral portion of
the moving yolk mass (that region about 4 mm from the egg
surface) was 4.1±0.5 mm/minute, while those approximately 6
mm from the cell surface moved at an average velocity of
8.1±0.8 mm/minute. (The deviation reported for each of these
figures represents the variability among measurements
obtained from different yolk platelets in the same egg.)
Movement near the interface between the cortex and the cytoplasmic
core (at a depth of about 4 mm) was quite turbulent,
with stationary organelles being jostled by those moving past
them. Some smaller organelles in this interface zone exhibited
sporadic movements, with short bursts interrupted by quiescent
periods. At greater depths (8-14 mm inside the plasma
membrane), medium- to large-sized yolk platelets (>5 mm
diameter) were plentiful and tended to move more smoothly
than the small yolk platelets at shallower depths, so we used
them for all distance measurements at depths greater than 6
mm. In a zone approximately 8 mm deep, yolk platelets moved
at a peak velocity (during the translocation phase) averaging
10.7±0.2 mm/minute. Yolk platelets deeper than 8 mm moved
with an average velocity which was not significantly different
from that seen at 8 mm (Fig. 6).
Analyses performed with other eggs, both fertilized and
electrically activated, showed a similar increase of velocity
with depth. Velocities in other fertilized eggs (n=5) varied
from 5.0±1.0 mm/minute at a depth of 4 mm to 10.5±1.0
mm/minute at a depth of 8 mm. (The deviation reported for each
of these figures represents the variability among means
obtained from different eggs.) In activated eggs (n=3), we
found that velocity varied from 5.0±1.0 mm/minute at a depth
of 4 mm to 8.5±1.0 mm/minute at a depth of 8 mm, suggesting
that variation of velocity with depth may be slightly less pronounced
in activated eggs than in fertilized eggs.
To verify that the differences that we observed at various
depths were not optical artifacts produced by the apparatus, we
examined several fixed eggs as described in Materials and
Methods. We found that distance measurements made at each
depth in these controls were identical to within less than 1%,
C. A. Larabell and others
Fig. 3. Single optical sections
of the region shown in Fig. 2,
but at a later time. These
images show rapid yolk
platelet movement between
(A) 0.72 NT and (B) 0.77
NT. Platelets have traveled
an average of 23.5 mm in 3.1
minutes. Microtubule bundles
of the parallel array can be
seen as dark channels among
the brightly stained yolk
platelets. The microtubules
bundles, which in effect are
negatively stained, form distinctive patterns (such as the âXâ seen here) that can be followed through consecutive images. Specific platelets
appear to be consistently associated with specific sites on the bundles, confirming that the inner cytoplasm and the parallel array move together
during rotation. Bar, 30 mm.
Fig. 4. Single optical section of the same region (8 mm inside the
plasma membrane at the vegetal pole) in a fertilized, immobilized
egg which was both stained with Nile Red and injected with
fluorescein-labeled tubulin. The channels between the Nile Redstained
yolk platelets (red) are filled with fluorescein-labeled
microtubules (green), confirming that the channels are negatively
stained images of microtubule bundles. Optical sections were made
at the rhodamine and fluorescein wavelengths simultaneously, then
superimposed by computer. Bar, 50 mm.
Fig. 5.
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Fig. 3. Single optical sections
of the region shown in Fig. 2,
but at a later time. These
images show rapid yolk
platelet movement between
(A) 0.72 NT and (B) 0.77
NT. Platelets have traveled
an average of 23.5 mm in 3.1
minutes. Microtubule bundles
of the parallel array can be
seen as dark channels among
the brightly stained yolk
platelets. The microtubules
bundles, which in effect are
negatively stained, form distinctive patterns (such as the âXâ seen here) that can be followed through consecutive images. Specific platelets
appear to be consistently associated with specific sites on the bundles, confirming that the inner cytoplasm and the parallel array move together
during rotation. Bar, 30 mm.
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Fig. 4. Single optical section of the same region (8 mm inside the
plasma membrane at the vegetal pole) in a fertilized, immobilized
egg which was both stained with Nile Red and injected with
fluorescein-labeled tubulin. The channels between the Nile Redstained
yolk platelets (red) are filled with fluorescein-labeled
microtubules (green), confirming that the channels are negatively
stained images of microtubule bundles. Optical sections were made
at the rhodamine and fluorescein wavelengths simultaneously, then
superimposed by computer. Bar, 50 mm.
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Fig. 5. Optical section perpendicular to the plasma membrane (i.e.,
parallel to the animal-vegetal axis) at the vegetal pole of a fertilized,
immobilized egg during cortical rotation. The inner cytoplasm
(indicated here by brightly stained yolk platelets) is separated from
the plasma membrane by a region 4-5 mm thick which is devoid of
yolk platelets. The bright red line at the bottom is the coverslip,
which was coated with poly-L-lysine to insure firm adhesion of the
egg surface. Bar, 20 mm.
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Fig. 6. Velocity of yolk platelets at five different depths inside the plasma membrane (PM) at the vegetal pole of a fertilized, immobilized egg
during cortical rotation. No yolk platelets were detected in the region 0-4 mm inside the PM and confocal images deeper than 14 mm were too
faint to be analyzed. In this egg, the velocity of yolk platelets during the translocation period increases with depth from 4 to 8 mm, averaging
4.1±0.5 mm/minute at a depth of 4 mm, 8.1±0.8 mm/minute at 6 mm and 10.7±0.2 mm/minute at 8 mm. (The deviation reported for each of these
figures represents the variability among measurements obtained from different yolk platelets.)
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Fig. 7. Polymerization of rhodamine-labeled tubulin near the vegetal pole of a fertilized, immobilized egg, in a region 4-6 mm inside the plasma
membrane. Short segments of microtubules are first detected at approximately 0.40 NT (A). Microtubules increase in length and density until
0.54 NT (H). Displacement of yolk platelets in the direction of rotation (movement of black circles from lower left to upper right of each
image) can also be seen in these sections (arrows in C and D). Each image represents a single optical section. Normalized times are: A, 0.40; B,
0.42; C, 0.44; D, 0.46; E, 0.48; F, 0.50; G, 0.52; H, 0.54. Bar, 20 mm.
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Fig. 8. Series of single
optical sections near the
vegetal pole of a fertilized,
immobilized egg at 0.65
NT, previously injected
with rhodamine-labeled
tubulin. The first image
(A) shows microtubules
approximately 4 mm inside
the plasma membrane;
each successive image is 1
mm deeper. Due to the
spherical shape of the egg,
the microtubules visible at
the periphery of the
deepest sections actually
lie at shallower depths.
These images show that
the parallel array is densest
between 4 and 6 mm (AC),
and becomes less dense
with depth until it is no
longer detectable at depths
greater than 8 mm (H-I).
Bar, 100 mm.
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Fig. 9. Microtubule dynamics during cortical rotation. Optical sections collected very rapidly show lateral movements of microtubule bundles
in the parallel array of a fertilized, immobilized egg injected with rhodamine-labeled tubulin. The arrowhead in each frame points to a segment
of a microtubule bundle, which undergoes a wave-like lateral displacement of approximately 2 mm in an 18 second time interval. The black
dots are pigment granules (»0.25 mm diameter). Bar, 3 mm.
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