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Dev Biol
2000 Aug 15;2242:250-62. doi: 10.1006/dbio.2000.9773.
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Local inhibition of cortical rotation in Xenopus eggs by an anti-KRP antibody.
Marrari Y
,
Terasaki M
,
Arrowsmith V
,
Houliston E
.
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The dorsal-ventral axis of amphibian embryos is specified by the "cortical rotation," a translocation of the egg cortex relative to the vegetal yolk mass. The mechanism of cortical rotation is not understood but is thought to involve an array of aligned, commonly oriented microtubules. We have demonstrated an essential requirement for kinesin-related proteins (KRPs) in the cortical rotation by microinjection beneath the vegetal cortex of an antipeptide antibody recognising multiple Xenopus egg KRPs. Time-lapse videomicroscopy revealed a striking local inhibition of the cortical rotation around the injection site, indicating that KRP-mediated translocation of the cortex is generated by forces acting across the vegetal subcortical region. Anti-tubulin immunofluorescence showed that the antibody disrupted both formation and maintenance of the aligned microtubule array. Direct examination of rhodamine-labelled microtubules by confocal microscopy showed that the anti-KRP antibody provoked striking three-dimensional flailing movement of the subcortical microtubules. In contrast, microtubules in antibody-free regions undulated only within the plane of the cortex, a significant population exhibiting little or no net movement. These findings suggest that KRPs have a critical role during cortical rotation in tethering microtubules to the cortex and that they may not contribute significantly to the translocation force as previously thought.
FIG. 1. Xenopus egg KRPs recognised by anti-LAGSE. Samples of
Xenopus egg cytoplasmic proteins (cyto) and of a motor protein
preparation enriched in certain KRPs (KRP—see Materials and
Methods) were subjected to SDS–PAGE and then either stained
with Coomassie blue to reveal total polypeptides or transferred
electrophoretically to nitrocellulose for immunodetection with
anti-LAGSE, anti-HIPYR, or the anti-kinesin heavy chain antibody
SUK4 as indicated. The 130K band corresponding to Eg5 is indicated.
Eg5 was detected by specific antibodies in eggcytoplasm and
associates with microtubules (not shown), but was not released
under the elution conditions used here.
FIG. 2. Anti-LAGSE inhibits cortical rotation locally. (a) Image from a time-lapse video sequence showing DiOC6(3)-labelled
mitochondrial islands embedded in the vegetal cytoplasmic mass of a Xenopus egg. In all other images (b–i) the translocation of
mitochondrial islands with respect to the immobilised vegetal cortex during cortical rotation is demonstrated by averaging 16–20
consecutive images. Black arrows have been added to show the direction of translocation. White arrows indicate the positions of
fluorescent beads injected with the various antibody solutions beneath the vegetal cortex. (b) Translocation in the uninjected egg
shown in (a). (c) Egg injected with 3 3 5 nl of 2.8 mg/ml anti-HIPYR at 0.5 normalised time (NT; see Materials and Methods).
Translocation was barely perturbed. (d) Egg injected with 25 nl of 2 mg/ml anti-LAGSE at 0.5 NT. Mitochondrial island translocation
was perturbed locally around the injection site, with “downstream†mitochondrial islands tearing as they moved away from the
affected area. (e, f) Two eggs injected with 2 3 10 nl of 2 mg/ml anti-LAGSE at 0.6 NT showing extensive inhibition of translocation.
Movement was completely blocked over most of the field shown in (f), while in (e) cytoplasm can be seen “piling up†behind the
affected region. (g, h) Effect of pretreating anti-LAGSE with the LAGSE peptide. 2 3 10 nl of 2 mg/ml antibody was injected at 0.4 NT
in both eggs. Incubation with a 25 molar excess of peptide substantially reversed the inhibitory effect of anti-LAGSE (g), whereas
overnight incubation at 4°C did not reduce the effectiveness of the antibody alone (h). (i) Egg injected with 3 3 7 nl of 5 mg/ml SUK4
at 0.5 NT. Translocation was barely perturbed.
FIG. 3. Anti-LAGSE blocks cell division. (a, b) Vegetal subcortical
injection of anti-LAGSE blocked cleavage furrow progression when
the injected antibody was positioned appropriately. In the embryo
shown in (b) both first and second furrows failed to complete, while
an uninjected sibling (a) divided successfully to the four-cell stage.
(c–f) Blastula stage Xenopus embryos which were injected at the
eight-cell stage with anti-LAGSE (c, d) or anti HIPYR (e, f) (8–10 nl
at 2 or 2.8 mg/ml, respectively, into single blastomeres). The
progeny of the blastomeres injected with anti-LAGSE failed to
divide while those injected with anti HIPYR continued to divide in
parallel with uninjected blastomeres. Immunofluorescence with
anti-tubulin antibodies (c, e) and anti-rabbit Ig antibodies to reveal
the injected antibody (d, f) showed that division of nuclei and
centrosomes in blastomeres containing anti-LAGSE antibody continued
in a disorganised way without cleavage while blastomeres in
sibling embryos containing anti-HIPYR showed normal interphase
microtubule arrays and spindles.
FIG. 4. Disruption of microtubule organisation by anti-LAGSE.
Microtubule organisation in eggs injected with 2 3 10 nl of 2
mg/ml anti-LAGSE at 0.43 NT (a– c), of 2 mg/ml anti-LAGSE at
0.58 NT (d, e), of 2.8 mg/ml anti-HIPYR at 0.43 NT (f), and of 2
mg/ml anti-LAGSE at 0.55 NT (g) and in an uninjected egg (h). All
eggs were fixed towards the end of the rotation period (0.7– 0.8 NT).
(a–f) Superimposed confocal images taken from the vegetal side of
eggs processed for immunofluorescence with anti-tubulin antibodies
(red) to show the distribution of microtubules and with antirabbit
Ig antibodies (green) to reveal the location of the injected
antibody. (a– c) Different vegetal regions of a single egg. Alignment
of the subcortical microtubules was normal in regions lacking
anti-LAGSE (a) but was progressively disrupted at increasing antibody
concentrations (b, c). In areas where the antibody was most
concentrated (c) only short microtubule segments were visible at
the cortex (arrows). Anti-LAGSE also provoked disruption of microtubules
in a dose-dependent manner when injected after the
formation of the array (d, e). In contrast, anti-HIPYR had little or no
appreciable effect on the organisation of the subcortical microtubules,
whether injected before (f) or during (not shown) the cortical
rotation. (g, h) Composite images constructed using the projection
function in NIH Image software from stacks of six to eight images
taken at 0.8-mm intervals downwards from the vegetal surface. For
each egg, the stacks have been tilted by 40° with respect to a
horizontal plane to show the upper (g, h) and lower (g9, h9) sides.
Microtubules projecting from the deeper cytoplasm towards the
surface (arrows) were clearly detectable in the presence of anti-
LAGSE (g) as in uninjected eggs (h); however, these failed to turn
and organise parallel to the cortex. This contributed to the severe
disorganisation of the subcortical microtubule array provoked by
the antibody. In contrast microtubules arriving at the surface in
uninjected eggs (h, h9) turned to join bundles of aligned microtubules
running parallel to the surface (for example at arrows).
FIG. 5. Anti-LAGSE uncouples microtubule–cortex interactions. Confocal images from sequences of three regions taken from the vegetal side
of a live prick-activated egg injected with 40 nl of 5 mg/ml rhodamine–tubulin 20 min after activation and 2 3 20 nl of 2 mg/ml anti-LAGSE 30
min later. Yolk platelets can be observed in the recording as unstained objects moving across the field, lying mainly in a deeper focal plane than
the microtubules. (a) Region far from the antibody injection site. Coordinated translocation of vegetal yolk platelets occurred undisturbed from
right to left relative to the immobilised cortex. The microtubules were well aligned and microtubule waves tended to move in the plane of the
cortex in the same direction as the yolk (e.g., at white arrows: the initial position is also marked lightly on the final image). Many microtubule
segments, however, showed little apparent movement (black arrow). (b) Region on the edge of the injected zone. On the right side of this field
the speed of yolk platelet translocation was reduced and the alignment of microtubules disrupted, with exaggerated lateral waving occurring
(arrows). (c) Region close to the antibody injection site where yolk platelet translocation was abolished. Microtubule behaviour was severely
disrupted. No net microtubule displacement occurred within the field; however, uncoordinated flailing of microtubule bundles was observed
both in the plane of the cortex and perpendicular to it (e.g., at arrows). The end of the bundle marked with a black arrow showed oscillating lateral
movements but clearly did not move from the field of observation.
FIG. 6. KRPs colocalise with ER in the cortical region. (a– d) Double immunofluorescence of eggs fixed during the cortical rotation with
anti-tubulin (a, c) and anti-LAGSE (b, d). Anti-LAGSE showed a carpet of superficial cortical staining (a, b) as well as a general codistribution
with the more deeply bundled microtubules of the subcortical vegetal microtubule array (c, d). (e– h) Double immunofluorescence with
anti-LAGSE and anti-BiP to reveal the ER. The distribution of ER at cortical (e) and subcortical (f) levels was similar to that of anti-LAGSE.
Superimposition of images taken both levels (g, h) at high magnification, with the ER outlined in red by application of an edge-finding
function to the anti-BiP image and anti-LAGSE in green, confirmed that the dots of anti-LAGSE overlay parts of the ER network (arrows).
FIG. 7. Possible mechanism for the cortical rotation. This diagram shows one possible mechanism which could account for our various
observations. Cortically attached KRPs (blue) and cytoplasmic minus-end-directed microtubule motors (green) cooperate to produce the
cortical rotation. Microtubules are indicated in red and the cortex in black. KRPs, perhaps associated with cortical ER, mediate lateral
interactions between outward-extending microtubules and the cortex. The tethered subcortical microtubules are drawn further into
alignment as the cortex moves (Vincent et al., 1987; Zisckind and Elinson, 1990), with movement and alignment probably reinforcing each
other (Gerhart et al., 1989). In this model, minus-end-directed motors drive microtubules out from the inner cytoplasm and exert a force
that displaces the cortex (green arrowheads).
