Patterns of microtubule polymerization relating to cortical rotation in Xenopus laevis eggs

Development ◽  
1991 ◽  
Vol 112 (1) ◽  
pp. 107-117 ◽  
Author(s):  
E. Houliston ◽  
R.P. Elinson
Keyword(s):  
Cortical Microtubule ◽  
Microtubule Polymerization ◽  
Vegetal Cortex ◽  
Sperm Entry ◽  
Female Pronucleus ◽  
Cortical Array ◽  
Xenopus Egg ◽  
Cytoplasmic Microtubules ◽  
Tubulin Immunofluorescence ◽  
Sperm Aster

Following fertilization, the Xenopus egg cortex rotates relative to the cytoplasm by 30 degrees about a horizontal axis. The direction of rotation, and as a result the orientation of the embryonic body axes, is normally specified by the position of sperm entry. The mechanism of rotation appears to involve an array of aligned microtubules in the vegetal cortex (Elinson and Rowning, 1988, Devl Biol. 128, 185–197). We performed anti-tubulin immunofluorescence on sections to follow the formation of this array. Microtubules disappear rapidly from the egg following fertilization, and reappear first in the sperm aster. Surprisingly, astral microtubules then extend radially through both the animal and vegetal cytoplasm. The cortical array arises as they reach the vegetal cell surface. The eccentric position of the sperm aster gives asymmetry to the formation of the array and may explain its alignment since microtubules reaching the cortex tend to bend away from the sperm entry side. The radial polymerization of cytoplasmic microtubules is not dependent on the sperm aster or on the female pronucleus: similar but more symmetric patterns arise in artificially activated and enucleate eggs, slightly later than in fertilized eggs. These observations suggest that the cortical microtubule array forms as a result of asymmetric microtubule growth outward from cytoplasm to cortex and, since cortical and cytoplasmic microtubules remain connected throughout the period of the rotation, that the microtubules of the array rotate with the cytoplasm.

Organization and regulation of cortical microtubules during the first cell cycle of Xenopus eggs

Development ◽  
1992 ◽  
Vol 114 (3) ◽  
pp. 699-709 ◽  
Author(s):  
M.M. Schroeder ◽  
D.L. Gard
Keyword(s):  
Cell Cycle ◽  
Cortical Microtubule ◽  
Cortical Microtubules ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence ◽  
Vegetal Cortex ◽  
M Phase ◽  
Tubulin Antibodies ◽  
Microtubule Networks ◽  
Sperm Aster

Anti-tubulin antibodies and confocal immunofluorescence microscopy were used to examine the organization and regulation of cytoplasmic and cortical microtubules during the first cell cycle of fertilized Xenopus eggs. Appearance of microtubules in the egg cortex temporally coincided with the outgrowth of the sperm aster. Microtubules of the sperm aster first reached the animal cortex at 0.25, (times normalized to first cleavage), forming a radially organized array of cortical microtubules. A disordered network of microtubules was apparent in the vegetal cortex as early as 0.35. Cortical microtubule networks of both animal and vegetal hemispheres were reorganized at times corresponding to the cortical rotation responsible for specification of the dorsal-ventral (D-V) axis. Optical sections suggest that the cortical microtubules are continuous with the microtubules of the sperm aster in fertilized eggs, or an extensive activation aster in activated eggs. Neither assembly and organization, nor disassembly of the cortical microtubules coincided with MPF activation during mitosis. However, cycloheximide or 6-dimethylaminopurine, which arrest fertilized eggs at interphase, blocked cortical microtubule disassembly. Injection of p13, a protein that specifically inhibits MPF activation, delayed or inhibited cortical microtubule breakdown. In contrast, eggs injected with cyc delta 90, a truncated cyclin that arrest eggs in M-phase, showed normal microtubule disassembly. Finally, injection of partially purified MPF into cycloheximide-arrested eggs induced cortical microtubule breakdown. These results suggest that, despite a lack of temporal coincidence, breakdown of the cortical microtubules is dependent on the activation of MPF.

scholarly journals Establishment of Polarity during Organization of the Acentrosomal Plant Cortical Microtubule Array

2006 ◽  
Vol 17 (3) ◽  
pp. 1298-1305 ◽  
Author(s):  
Ram Dixit ◽  
Eric Chang ◽  
Richard Cyr
Keyword(s):  
Fluorescence Recovery After Photobleaching ◽  
Cortical Microtubule ◽  
Cortical Microtubules ◽  
Predominant Role ◽  
Microtubule Array ◽  
Ordered Arrays ◽  
Microtubule Polymerization ◽  
Selective Stabilization ◽  
Cortical Array ◽  
The Relationship

The plant cortical microtubule array is a unique acentrosomal array that is essential for plant morphogenesis. To understand how this array is organized, we exploited the microtubule (+)-end tracking activity of two Arabidopsis EB1 proteins in combination with FRAP (fluorescence recovery after photobleaching) experiments of GFP-tubulin to examine the relationship between cortical microtubule array organization and polarity. Significantly, our observations show that the majority of cortical microtubules in ordered arrays, within a particular cell, face the same direction in both Arabidopsis plants and cultured tobacco cells. We determined that this polar microtubule coalignment is at least partially due to a selective stabilization of microtubules, and not due to a change in microtubule polymerization rates. Finally, we show that polar microtubule coalignment occurs in conjunction with parallel grouping of cortical microtubules and that cortical array polarity is progressively enhanced during array organization. These observations reveal a novel aspect of plant cortical microtubule array organization and suggest that selective stabilization of dynamic cortical microtubules plays a predominant role in the self-organization of cortical arrays.

scholarly journals An ultrastructural study of cross-fertilization (Arbacia female x Mytilus male).

1977 ◽  
Vol 73 (1) ◽  
pp. 14-26 ◽  
Author(s):  
F J Longo
Keyword(s):  
Sea Urchin ◽  
Ultrastructural Study ◽  
Cortical Granule ◽  
Male Pronucleus ◽  
Female Pronucleus ◽  
Sperm Nuclei ◽  
Cross Fertilization ◽  
Sperm Aster

Insemination of sea urchin (Arbacia) ova with mussel (Mytilus) sperm has been accomplished by treating eggs with trypsin and suspending the gametes in seawater made alkaline with NaOH. Not all inseminated eggs undergo a cortical granule reaction. Some eggs either elevate what remains of their vitelline layer or demonstrate no cortical modification whatsoever. After its incorporation into the egg, the nucleus of Mytilus sperm undergoes changes which eventually give rise to the formation of a male pronucleus. Concomitant with these transformations, a sperm aster may develop in association with the centrioles brought into the egg with the spermatozoon. Both the male pronucleus and the sperm aster may then migrate centrad to the female pronucleus. Evidence is presented which suggests that fusion of the male pronuclei from Mytilus sperm with female pronuclei from Arbacia eggs may occur, although this was not directly observed. These results demonstrate that Mytilus sperm nuclei are able to react to conditions within Arbacia eggs and differentiate into male pronuclei.

Fertilization and ooplasmic movements in the ascidian egg

Development ◽  
1989 ◽  
Vol 105 (2) ◽  
pp. 237-249 ◽  
Author(s):  
C. Sardet ◽  
J. Speksnijder ◽  
S. Inoue ◽  
L. Jaffe
Keyword(s):  
Polar Body ◽  
Surface Velocity ◽  
Second Phase ◽  
Animal Pole ◽  
Male Pronucleus ◽  
Vegetal Pole ◽  
Female Pronucleus ◽  
Ooplasmic Segregation ◽  
Second Polar Body ◽  
Sperm Aster

Using light microscopy techniques, we have studied the movements that follow fertilization in the denuded egg of the ascidian Phallusia mammillata. In particular, our observations show that, as a result of a series of movements described below, the mitochondria-rich subcortical myoplasm is split in two parts during the second phase of ooplasmic segregation. This offers a potential explanation for the origin of larval muscle cells from both posterior and anterior blastomeres. The first visible event at fertilization is a bulging at the animal pole of the egg, which is immediately followed by a wave of contraction, travelling towards the vegetal pole with a surface velocity of 1.4 microns s-1. This wave accompanies the first phase of ooplasmic segregation of the mitochondria-rich subcortical myoplasm. After this contraction wave has reached the vegetal pole after about 2 min, a transient cytoplasmic lobe remains there until 6 min after fertilization. Several new features of the morphogenetic movements were then observed: between the extrusion of the first and second polar body (at 5 and 24–29 min, respectively), a series of transient animal protrusions form at regular intervals. Each animal protrusion involves a flow of the centrally located cytoplasm in the animal direction. Shortly before the second polar body is extruded, a second transient vegetal lobe (‘the vegetal button’) forms, which, like the first, resembles a protostome polar lobe. Immediately after the second polar body is extruded, three events occur almost simultaneously: first, the sperm aster moves from the vegetal hemisphere to the equator. Second, the bulk of the vegetally located myoplasm moves with the sperm aster towards the future posterior pole, but interestingly about 20% remains behind at the anterior side of the embryo. This second phase of myoplasmic movement shows two distinct subphases: a first, oscillatory subphase with an average velocity of about 6 microns min-1, and a second steady subphase with a velocity of about 26 microns min-1. The myoplasm reaches its final position as the male pronucleus with its surrounding aster moves towards the centre of the egg. Third, the female pronucleus moves towards the centre of the egg to meet with the male pronucleus. Like the myoplasm, the migrations of both the sperm aster and the female pronucleus shows two subphases with distinctly different velocities. Finally, the pronuclear membranes dissolve, a small mitotic spindle is formed with very large asters, and at about 60–65 min after fertilization, the egg cleaves.

scholarly journals The Saccharomyces cerevisiae Kinesin-related Motor Kar3p Acts at Preanaphase Spindle Poles to Limit the Number and Length of Cytoplasmic Microtubules

1997 ◽  
Vol 137 (2) ◽  
pp. 417-431 ◽  
Author(s):  
William Saunders ◽  
David Hornack ◽  
Valerie Lengyel ◽  
Changchun Deng
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Body Region ◽  
Microtubule Depolymerization ◽  
Growth Defects ◽  
Microtubule Polymerization ◽  
Late Anaphase ◽  
A Cell ◽  
Cytoplasmic Microtubules

The Saccharomyces cerevisiae kinesin-related motor Kar3p, though known to be required for karyogamy, plays a poorly defined, nonessential role during vegetative growth. We have found evidence suggesting that Kar3p functions to limit the number and length of cytoplasmic microtubules in a cell cycle–specific manner. Deletion of KAR3 leads to a dramatic increase in cytoplasmic microtubules, a phenotype which is most pronounced from START through the onset of anaphase but less so during late anaphase in synchronized cultures. We have immunolocalized HA-tagged Kar3p to the spindle pole body region, and fittingly, Kar3p was not detected by late anaphase. A microtubule depolymerizing activity may be the major vegetative role for Kar3p. Addition of the microtubule polymerization inhibitors nocodazol or benomyl to the medium or deletion of the nonessential α-tubulin TUB3 gene can mostly correct the abnormal microtubule arrays and other growth defects of kar3 mutants, suggesting that these phenotypes result from excessive microtubule polymerization. Microtubule depolymerization may also be the mechanism by which Kar3p acts in opposition to the anaphase B motors Cin8p and Kip1p. A preanaphase spindle collapse phenotype of cin8 kip1 mutants, previously shown to involve Kar3p, is markedly delayed when microtubule depolymerization is inhibited by the tub2-150 mutation. These results suggest that the Kar3p motor may act to regulate the length and number of microtubules in the preanaphase spindle.

scholarly journals Microtubule dynamics at the G2/M transition: abrupt breakdown of cytoplasmic microtubules at nuclear envelope breakdown and implications for spindle morphogenesis.

1996 ◽  
Vol 135 (1) ◽  
pp. 201-214 ◽  
Author(s):  
Y Zhai ◽  
P J Kronebusch ◽  
P M Simon ◽  
G G Borisy
Keyword(s):  
Nuclear Envelope ◽  
Fluorescence Ratio ◽  
Subsequent Increase ◽  
Abrupt Decrease ◽  
Nuclear Envelope Breakdown ◽  
Anaphase Onset ◽  
Chromosome Attachment ◽  
Direct Fluorescence ◽  
Cytoplasmic Microtubules ◽  
Tubulin Immunofluorescence

We recently developed a direct fluorescence ratio assay (Zhai, Y., and G.G. Borisy. 1994. J. Cell Sci. 107:881-890) to quantify microtubule (MT) polymer in order to determine if net MT depolymerization occurred upon anaphase onset as the spindle was disassembled. Our results showed no net decrease in polymer, indicating that the disassembly of kinetochore MTs was balanced by assembly of midbody and astral MTs. Thus, the mitosis-interphase transition occurs by a redistribution of tubulin among different classes of MTs at essentially constant polymer level. We now examine the reverse process, the interphase-mitosis transition. Specifically, we quantitated both the level of MT polymer and the dynamics of MTs during the G2/M transition using the fluorescence ratio assay and a fluorescence photoactivation approach, respectively. Prophase cells before nuclear envelope breakdown (NEB) had high levels of MT polymer (62%) similar to that previously reported for random interphase populations (68%). However, prophase cells just after NEB had significantly reduced levels (23%) which recovered as MT attachments to chromosomes were made (prometaphase, 47%; metaphase, 56%). The abrupt reorganization of MTs at NEB was corroborated by anti-tubulin immunofluorescence staining using a variety of fixation protocols. Sensitivity to nocodazole also increased at NEB. Photoactivation analyses of MT dynamics showed a similar abrupt change at NEB, basal rates of MT turnover (pre-NEB) increased post-NEB and then became slower later in mitosis. Our results indicate that the interphase-mitosis (G2/M) transition of the MT array does not occur by a simple redistribution of tubulin at constant polymer level as the mitosis-interphase (M/G1) transition. Rather, an abrupt decrease in MT polymer level and increase in MT dynamics occurs tightly correlated with NEB. A subsequent increase in MT polymer level and decrease in MT dynamics occurs correlated with chromosome attachment. These results carry implications for understanding spindle morphogenesis. They indicate that changes in MT dynamics may cause the steady-state MT polymer level in mitotic cells to be lower than in interphase. We propose that tension exerted on the kMTs may lead to their lengthening and thereby lead to an increase in the MT polymer level as chromosomes attach to the spindle.

scholarly journals A plausible mechanism for longitudinal lock-in of cortical microtubule array after light-induced reorientation

2020 ◽  
Author(s):  
Marco Saltini ◽  
Bela M. Mulder
Keyword(s):  
Alternative Hypothesis ◽  
Cortical Microtubule ◽  
Selective Advantage ◽  
Consensus Model ◽  
Microtubule Array ◽  
Orientation State ◽  
Cortical Array ◽  
Plausible Mechanism ◽  
Lock In

The light-induced reorientation of the cortical microtubule array in dark-grown A. thaliana hypocotyl cells is a striking example of the dynamical plasticity of the microtubule cytoskeleton. A consensus model, based on katanin-mediated severing at microtubule crossovers, has been developed that successfully describes the onset of the observed switch between a transverse and longitudinal array orientation. However, we currently lack an understanding of of why the newly populated longitudinal array direction remains stable for longer times, when the initial trigger for the reorientation has died out, and re-equilibration effects would tend to drive the system back to a mixed orientation state. Using both simulations and analytical calculations, we show that the assumption of a small orientation-dependent shift in microtubule dynamics is sufficient to explain the long term lock-in of the longitudinal array orientation. Furthermore, we show that the natural alternative hypothesis that there is a selective advantage in severing longitudinal microtubules, is neither necessary nor sufficient to achieve cortical array reorientation, but is able to accelerate this process significantly.

scholarly journals Polarity and reorganization of the endoplasmic reticulum during fertilization and ooplasmic segregation in the ascidian egg.

1993 ◽  
Vol 120 (6) ◽  
pp. 1337-1346 ◽  
Author(s):  
J E Speksnijder ◽  
M Terasaki ◽  
W J Hage ◽  
L F Jaffe ◽  
C Sardet
Keyword(s):  
Thick Layer ◽  
Second Phase ◽  
Calcium Waves ◽  
Cell Region ◽  
Vegetal Cortex ◽  
Before And After ◽  
Ooplasmic Segregation ◽  
Two Phases ◽  
Sperm Aster

During the first cell cycle of the ascidian egg, two phases of ooplasmic segregation create distinct cytoplasmic domains that are crucial for later development. We recently defined a domain enriched in ER in the vegetal region of Phallusia mammillata eggs. To explore the possible physiological and developmental function of this ER domain, we here investigate its organization and fate by labeling the ER network in vivo with DiIC16(3), and observing its distribution before and after fertilization in the living egg. In unfertilized eggs, the ER-rich vegetal cortex is overlaid by the ER-poor but mitochondria-rich subcortical myoplasm. Fertilization results in striking rearrangements of the ER network. First, ER accumulates at the vegetal-contraction pole as a thick layer between the plasma membrane and the myoplasm. This accompanies the relocation of the myoplasm toward that region during the first phase of ooplasmic segregation. In other parts of the cytoplasm, ER becomes progressively redistributed into ER-rich and ER-poor microdomains. As the sperm aster grows, ER accumulates in its centrosomal area and along its astral rays. During the second phase of ooplasmic segregation, which takes place once meiosis is completed, the concentrated ER domain at the vegetal-contraction pole moves with the sperm aster and the bulk of the myoplasm toward the future posterior side of the embryo. These results show that after fertilization, ER first accumulates in the vegetal area from which repetitive calcium waves are known to originate (Speksnijder, J. E. 1992. Dev. Biol. 153:259-271). This ER domain subsequently colocalizes with the myoplasm to the presumptive primary muscle cell region.

Provisional bilateral symmetry in Xenopus eggs is estiblished during maturation

Zygote ◽  
1994 ◽  
Vol 2 (3) ◽  
pp. 213-220 ◽  
Author(s):  
E.E. Brown ◽  
K.M. Margelot ◽  
M.V. Danilchik
Keyword(s):  
Cell Cycle ◽  
Bilateral Symmetry ◽  
Entry Point ◽  
Yolk Mass ◽  
Vegetal Cortex ◽  
Sperm Entry ◽  
Egg Cortex ◽  
The Way

SummaryDorsal–ventral patterning in the Xenopus egg becomes established midway through the first cell cycle during a 30° rotation of the subcortical yolk mass relative to the egg cortex. This rotation of symmetrisation is microtubule dependent, and its direction is thought to be cued by the usually eccentric sperm centrosome. The fact that parthenogenetically activated eggs also undergo a directed rotation, despite the absence of a sperm centrosome, suggests that an endogenous asymmetry in the unfertilised egg supports the directed polymerisation of microtubules in the vegetal cortex, in the way that an eccentric sperm centrosome would in fertilised eggs. Consistent with this idea, we noticed that the maturation spot is usually located an average of more than 15° from the geometric centre of the pigmented animal hemisphere. In parthenogenetically activated eggs, this eccentric maturation spot can be used to predict the direction of rotation. Although in most fertilised eggs the yolk mass rotates toward the sperm entry point (SEP) meridian, occasionally this relationship is perturbed significantly; in such eggs, the maturation spot is never on the same side of the egg as the SEP. In oocytes tilted 90° from upright during maturation in vitro, the maturation spot developed 15° or more from the centre of the pigmented hemisphere, always displaced towards the point on the equator that was up during maturation. This experimentally demonstrated lability is consistent with an off-axis oocyte orientation during oogenesis determining its eccentric maturation spot position, and, in turn, its endogenous rotational bias.

Analysis of microtubule polymerization in vitro and during the cell cycle in Xenopus egg extracts

Methods ◽  
2006 ◽  
Vol 38 (1) ◽  
pp. 29-34 ◽  
Author(s):  
Priya Prakash Budde ◽  
Arshad Desai ◽  
Rebecca Heald
Keyword(s):  
Cell Cycle ◽  
Microtubule Polymerization ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts
Sign in / Sign up

Export Citation Format

Share Document