Cortical Contraction Research Articles

A novel role for clathrin in cytokinesis.

Using clathrin-minus Dictyostelium cells, we identified a novel requirement for clathrin during cytokinesis. In suspension culture, clathrin-minus cells failed to divide and became large and multinucleate. This cytokinesis deficiency was not attributable to a pleiotropic effect on the actomyosin cytoskeleton, since other cellular events driven by myosin II (e.g., cortical contraction and capping of concanavalin A receptors) remained intact in clathrin-minus cells. Examination of cells expressing myosin II tagged with green fluorescent protein showed that clathrin-minus cells failed to assemble myosin II into a functional contractile ring. This inability to localize myosin II to a particular location was specific for cytokinesis, since clathrin-minus cells moving across a substrate localized myosin II properly to their posterior cortexes. These results demonstrate that clathrin is essential for construction of a functional contractile ring during cell division.

Microtubule-entrained kinase activities associated with the cortical cytoskeleton during cytokinesis.

Research over the past few years has demonstrated the central role of protein phosphorylation in regulating mitosis and the cell cycle. However, little is known about how the mechanisms regulating the entry into mitosis contribute to the positional and temporal regulation of the actomyosin-based contractile ring formed during cytokinesis. Recent studies implicate p34cdc2 as a negative regulator of myosin II activity, suggesting a link between the mitotic cycle and cytokinesis. In an effort to study the relationship between protein phosphorylation and cytokinesis, we examined the in vivo and in vitro phosphorylation of actin-associated cortical cytoskeletal (CSK) proteins in an isolated model of the sea urchin egg cortex. Examination of cortices derived from eggs or zygotes labeled with 32P-orthophosphate reveals a number of cortex-associated phosphorylated proteins, including polypeptides of 20, 43 and 66 kDa. These three major phosphoproteins are also detected when isolated cortices are incubated with [32P]ATP in vitro, suggesting that the kinases that phosphorylate these substrates are also specifically associated with the cortex. The kinase activities in vivo and in vitro are stimulated by fertilization and display cell cycle-dependent activities. Gel autophosphorylation assays, kinase assays and immunoblot analysis reveal the presence of p34cdc2 as well as members of the mitogen-activated protein kinase family, whose activities in the CSK peak at cell division. Nocodazole, which inhibits microtubule formation and thus blocks cytokinesis, significantly delays the time of peak cortical protein phosphorylation as well as the peak in whole-cell histone H1 kinase activity. These results suggest that a key element regulating cortical contraction during cytokinesis is the timing of protein kinase activities associated with the cortical cytoskeleton that is in turn regulated by the mitotic apparatus.

Activation ofXenopusEggs by RGD-Containing Peptides Accompanied by Intracellular Ca2+Release

To investigate the mechanism of egg activation inXenopus,unfertilized eggs were treated with various oligopeptides related to the RGD sequence-containing peptides. When dejellied eggs were treated with RGDS at concentrations higher than 250 μM,the eggs produced a positive-going activation potential concomitant with intracellular Ca2+release. The eggs underwent cortical granule breakdown, cortical contraction, and resumption of meiosis. The activation by RGDS was independent of the voltage of egg membranes. GRGD, GRGDTP, and RGPFPI caused activation in some eggs, but RGES, RFDS, GPRP amide, and GPA did not. Denuded eggs were activated by Sepharose beads bound to RGDS, indicating an interaction of the RGD sequence with receptor molecules on the egg plasma membrane. The activation by RGDS did not occur under a Ca2+- and Mg2+-free condition, but occurred in a Ca2+-free solution containing Mg2+, concomitant with an intracellular Ca2+release. These results indicate that a receptor of the RGD sequence on egg plasma membrane causes egg activation through an intracellular signal transduction system. A sperm agonist including the RGD sequence may play an important role in egg activation through the egg membrane receptor during the normal fertilization process.

Biochemical kinetic characterization of the Acanthamoeba myosin-I ATPase.

Acanthamoeba myosin-IA and myosin-IB are single-headed molecular motors that may play an important role in membrane-based motility. To better define the types of motility that myosin-IA and myosin IB can support, we determined the rate constants for key steps on the myosin-I ATPase pathway using fluorescence stopped-flow, cold-chase, and rapid-quench techniques. We determined the rate constants for ATP binding, ATP hydrolysis, actomyosin-I dissociation, phosphate release, and ADP release. We also determined equilibrium constants for myosin-I binding to actin filaments, ADP binding to actomyosin-I, and ATP hydrolysis. These rate constants define an ATPase mechanism in which (a) ATP rapidly dissociates actomyosin-I, (b) the predominant steady-state intermediates are in a rapid equilibrium between actin-bound and free states, (c) phosphate release is rate limiting and regulated by heavy-chain phosphorylation, and (d) ADP release is fast. Thus, during steady-state ATP hydrolysis, myosin-I is weakly bound to the actin filament like skeletal muscle myosin-II and unlike the microtubule-based motor kinesin. Therefore, for myosin-I to support processive motility or cortical contraction, multiple myosin-I molecules must be specifically localized to a small region on a membrane or in the actin-rich cell cortex. This conclusion has important implications for the regulation of myosin-I via localization through the unique myosin-I tails. This is the first complete transient kinetic characterization of a member of the myosin superfamily, other than myosin-II, providing the opportunity to obtain insights about the evolution of all myosin isoforms.

Transient effects of microgravity on early embryos of Xenopus laevis

In order to study the role of gravity on the early development of the clawed toad Xenopus laevis, we performed an experiment on the Maser-6 sounding rocket launched from Kiruna (Sweden) on 4 Nov 1993. The aim was to find out whether a short period of microgravity (μg) during fertilization and the first few minutes of development does indeed result in abnormal axis formation as was suggested by a pilot experiment on the Maser 3 in 1989. On the Maser 6 we used two new technical additions in the Fokker CIS unit, viz. a 1-g control centrifuge and a video recording unit which both worked successfully. The 1-g control centrifuge was used to discriminate between the influences of flight perturbations and μg. After fertilization shortly before launch, one of the first indications of successful egg activation, the cortical contraction, was registered in μg and on earth. Analysis of the video tapes revealed that the cortical contraction in μg starts earlier than at 1 g on earth. After recovery of the eggs fertilized in μg and culture of the embryos on earth, the morphology of the blastocoel has some consistent differences from blastulae from eggs fertilized in the 1-g centrifuge of the rocket. However from the gastrula stage onward, the μg embryos apparently recover and resume normal development: the XBra gene is normally expressed, and histological examination shows normal axis formation.

The sperm entry point defines the orientation of the calcium-induced contraction wave that directs the first phase of cytoplasmic reorganization in the ascidian egg

Ascidians eggs are spawned with their cytoskeleton and organelles organized along a preexisting animal-vegetal axis. Fertilization triggers a spectacular microfilament-dependant cortical contraction that causes the relocalization of preexisting cytoplasmic domains and the creation of new domains in the lower part of the vegetal hemisphere. We have investigated the relationship between fertilization, the cortical contraction and the localization of cytoplasmic domains in eggs of the ascidian Phallusia mammillata. We have also examined the link between this first phase of ooplasmic segregation and the site of gastrulation. The cortical contraction was found to be initiated on the side of the egg where intracellular calcium is first released either by the entering sperm or by photolysis of caged InsP3. The cortical contraction carries the sperm nucleus towards the vegetal hemisphere along with a subcortical mitochondria-rich domain (the myoplasm). If the sperm enters close to the animal or vegetal poles the cortical contraction is symmetrical, travelling along the animal-vegetal axis. If the sperm enters closer to the equator, the contraction is asymmetrical and its direction does not coincide with the animal-vegetal axis. The direction of contraction defines an axis along which preexisting (such as the myoplasm) or newly created cytoplasmic domains are relocalized. Two microfilament-rich surface constrictions, the 'contraction pole' and the 'vegetal button' (which forms 20 minutes later), appear along that axis approximately opposite the site where the contraction is initiated. The contraction pole can be situated as much as 55 degrees from the vegetal pole, and its location predicts the site of gastrulation. It thus appears that in ascidian eggs, the organization of the egg before fertilization defines a 110 degrees cone centered around the vegetal pole in which the future site of gastrulation of the embryo will lie. The calcium wave and cortical contraction triggered by the entering sperm adjust the location of cytoplasmic domains along an axis within that permissive zone. We discuss the relation between that axis and the establishment of the dorsoventral axis in the ascidian embryo.

Activation of Xenopus Eggs by Cynops Sperm Extract is Dependent upon Both Extra-and Intra-Cellular Ca Activities

Abstract When unfertilized Xenopus eggs were treated by Cynops sperm extract in 10% Steinberg's solution (SB), egg's membranes hyperpolarized to about −37 mV and then depolarized to elicit a positive-going potential amounting to about +34 mV. The eggs underwent cortical contraction and resumption of meiosis. Activation of eggs in various external solutions indicates that the hyperpolarization is due mainly to opening of Na channels, but the positive-going potential is due to Cl channels on the egg's plasma membranes. Since the activation was inhibited by CdCl2, CoCl2, or NiCl2 as well as by amiloride, Ca influx through Ca channels is necessary for the activation by the sperm extract. A propagative intracellular Ca release was induced not only by Cynops sperm, but also by their sperm extract. Injection of BAPTA or heparin into the eggs completely inhibited activation, indicating that egg activation requires an intracellular Ca release dependent upon receptors for inositol 1,4,5-trisphosphate.

Different calcium-dependent pathways control fertilisation-triggered glycoside release and the cortical contraction in ascidian eggs.

Fertilisation of ascidian eggs induces the rapid release of a cell surface N-acetylglycosaminidase that blocks sperm binding to vitelline coat sperm receptors resulting in a block to polyspermy. Fertilisation also triggers a large contraction of the egg (thus stimulating ooplasmic segregation) that is completed within 5 min of insemination. In eggs of the ascidian Phallusia mammillata, glycosidase release and cortical contractions are blocked by BAPTA-AM [bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester], a cell-permeant calcium chelator, indicating that both processes are probably dependent on a rise in intracellular calcium levels. Both glycosidase release and the cortical contraction are induced by treatment of the egg with the protein synthesis inhibitor emetine, while only the glycosidase release is induced by isoproterenol, carbachol or acetylcholine. Previous work with ryanodine demonstrated that ryanodine also caused glycosidase release but not the cortical contraction. Inversely, activation by ionomycin in calcium-free sea water causes cortical contractions but not glycosidase release. Thus the two processes can be activated independently. Dextran-coupled (10 kDa) calcium green-1 injected eggs show an increase in intracellular calcium 30-40 s before the cortical contraction is triggered by fertilisation or ionomycin-induced activation. This confirms previous findings that the cortical contraction is a consequence of the activation calcium wave triggered by the sperm. The extracellular calcium requirement for the glycosidase release suggests that calcium influx may be more important for this phase of egg activation. Thus activation of ascidian eggs appears to involve two independent pathways involving calcium.

Mechanically stimulated cytoskeleton rearrangement and cortical contraction in human neutrophils

A mechanical test with micropipets is used to characterize cytoskeleton rearrangement and contraction induced by mechanical stresses in human neutrophils. The yield shear resultant of the cell cortex is on the order of 0.06 to 0.09 mN.m-1. The measured yield shear resultant suggests that the neutrophil cortex is a weakly cross-linked structure. When a tether is pulled out from the cell surface, a polymer structure starts to fill it and spreads out from the cell body. The rate of advancement of the polymerization front is almost constant and, therefore, is not diffusion limited. The measured rate is much smaller than the one of spontaneous actin polymerization, suggesting that the limiting process is either the dissociation of actin monomers from their dimers with the capping proteins or the rate of formation of new nucleation sites or both. Polymerization is also observed after applying sufficient mechanical stresses on a small portion of the cell surface. The polymerization is followed by mass transfer from the cell into the prestressed region and later on by contraction of the main cell body. The pressure generating the flow is located in the prestressed region and most probably is a result of its "swelling" and contraction. The contraction of the main cell body is very similar (in its time dependence and magnitude) to the contraction during phagocytosis. The measured maximum cortical tension is on the order of 0.5 mN.m-1, which for a 3.5-microns diameter pipet corresponds to a maximum contraction force of 11 nN.

Pseudocleavage Is Dispensable for Polarity and Development in C. elegans Embryos

The first cleavage of the Caenorhabditis elegans embryo is asymmetrical, producing daughters with different cell fates. During the first cell cycle, P granules, cytoplasmic components that are segregated to the germ-line, are localized to the posterior of the embryo. It has been hypothesized that the asymmetrical behavior of the daughters of the first division results from a similar localization of developmental determinants. A process called pseudocleavage also occurs during the first cell cycle: Anterior cortical contractions culminate in a single partial constriction of the embryo called the pseudocleavage furrow. Coincident with pseudocleavage, there is an anteriorly directed flow of cortical cytoplasm and a posteriorly directed flow of internal cytoplasm. Foci of filamentous cortical actin become asymmetrically distributed into an anterior cap. Roles for these various first cell cycle events in cytoplasmic localization and development have been suggested but remain unclear. We have isolated a maternal effect mutation, nop-1 (it142), which abolishes the anterior cortical contractions and the pseudocleavage furrow. In addition, cortical actin foci remain uniformly distributed in most embryos. Despite these defects, cytoplasmic and cortical streaming is present and P granules are localized to the posterior of early embryos. In most embryos from mutant mothers, development proceeds normally and the embryos hatch and grow into fertile adults. We conclude that the pseudocleavage contractions and furrow are dispensable for the development of C. elegans.

Signal transduction during shrimp oocyte activation by extracellular Mg2+: roles of inositol 1,4,5-trisphosphate, tyrosine kinases and G-proteins

ABSTRACT Oocytes of the shrimp Sicyoniaingentis are naturally activated upon contact with seawater Mg2+. We investigated the mechanism of Mg2+-induced intracellular Ca2+ release and cortical contraction through treatment of oocytes with various activators and inhibitors of signal transduction pathways. Injection of oocytes with the second messenger inositol 1,4,5-trisphosphate resulted in an immediate rise in intracellular Ca2+ and normal cortical contraction. By contrast, injection of the GTP analog guanosine 5′-O-(3′-thiotriphosphate) to activate G-proteins did not affect intracellular Ca2+ levels but did induce cortical contraction. Tyrosine kinase inhibitors (tyrphostin and staurosporine) suppressed the Mg2+-induced Ca2+ rise and contraction, and the inhibition could be overcome by injection with inositol 1,4,5-trisphosphate. Immunoprecipitation of phosphotyrosine-containing proteins from oocyte lysates showed that a 110×103Mr protein was phosphorylated within seconds of oocyte exposure to Mg2+, and tyrphostin inhibited the phosphorylation of this protein. Pre-treatment of oocytes with the protease trypsin abolished their ability to release Ca2+ in response to extracellular Mg2+, indicating a role for a cell surface protein during normal oocyte activation; treated oocytes could be rescued by inositol 1,4,5-trisphosphate injection. These results suggest that S. ingentis oocytes are activated through a Mg2+ ‘receptor’ which activates a tyrosine kinase and results in the production of inositol 1,4,5-trisphosphate to release intracellular Ca2+ stores and induce cortical con-traction. A G-protein/GTPase may also be involved in the pathway leading to cortical contraction.

Development of cortical contractility in the Xenopus laevis oocyte mediated by reorganisation of the cortical cytoskeleton: a model.

As the amphibian oocyte becomes the fertilisation-competent egg an actin-myosin network assembles in the cortex which provides for the cortical contraction that accompanies fertilisation. A number of recent investigations provide data for development of a model detailing the structural changes which should accompany the development of this contractile network as well as the signalling mechanisms which regular assembly and contraction.

Cell migration by graded attachment to substrates and contraction

The migration of fibroblastic cells in vitro involves the extension of lamellipodia, attachment of the cytoskeleton through the plasma membrane to the extracellular matrix, and the generation of force that pulls attachments rearward and the cell forward. Bulk flow of membrane or lipid relative to the cell outline cannot be detected; however, crosslinked glycoproteins attached to the cytoskeleton move rearward and diffusing particles are driven forward by a motor mechanism. The leading edge is the preferential site for the cytoskeleton to attach to crosslinked glycoproteins including integrins. Force for moving the cell forward can be generated either by a cortical contraction acting as a net to pull the endoplasm forward or by motors at the boundary of the endoplasm and ectoplasm pulling on the cortical actin. As the cortical actin is anchored to the external matrix more strongly at the front of the cell than at the rear, contraction will pull the cell forward. Such a model has important implications for the nature of the glycoprotein attachments to the cytoskeleton and the regional differences in membrane structure.

Independent Initiation of Calcium Dependent Glycosidase Release and Cortical Contractions during the Activation of Ascidian Eggs: (Ascidian, Ryanodine, Thimerosal, Thapsigargin, N-acetylglucosaminidase).

During fertilization or ionophore induced activation, ascidian eggs rapidly release cell surface N-acetylglucosaminidase activity used in the block against polyspermy and undergo cortical contractions before they re-initiate meiosis. To better understand the activation process, we probed the relationship between these two processes in Ascidia ceratodes eggs by activating with different agents that increase intracellular Ca levels and under different ionic conditions. Glycosidase activity release was followed by the use of a fluorogenic substrate, and cortical contractions were followed by examining changes in cell shape with light microscopy. Ionomycin (2.7 μM) and thimerosal (1 mM) initiate glycosidase release and cortical contractions when administered in complete sea water (SW) but only the contractions in low Ca SW. Ryanodine (0.67 mM), known to raise free intracellular Ca in a number of cell types by release from the endoplasmic reticulum, causes glycosidase release but fails to initiate cortical contractions in complete SW. Thapsigargin (10 μM), which inhibits Ca dependent ATPase in the ER, causes glycosidase release but induces the contractions only about 50% of the time. These experiments show that, although glycosidase release normally precedes the ooplasmic shape changes that accompany the resumption of meiosis in ascidian eggs, they are not obligately coupled. That both processes can be induced by treatments known to raise intracellular Ca in other systems but under different conditions indicates that there may be a multiplicity of Ca requiring but functionally independent events during egg activation.

A genetically engineered, protein-based optical biosensor of myosin II regulatory light chain phosphorylation.

Myosin II is an important motor in the contraction of smooth and striated muscle as well as in a variety of non-muscle cell motile events including cytokinesis, cortical contractions during migration of fibroblasts, and capping of receptors. Phosphorylation of the 20-kDa light chain by myosin light chain kinase is part of the regulation of smooth muscle and mammalian nonmuscle myosin II. We designed, characterized, and tested the use of a protein-based optical biosensor to monitor this phosphorylation "switch." A regulatory light chain was genetically engineered to contain a single cysteine at amino acid position 18. The mutant light chain (Cys18.LC20), reacted with the fluorophore acrylodan, responded to phosphorylation of serine 19 with a fluorescence emission quenching of 60% and a 28-nm red-shift. When the acrylodan-labeled mutant light chain (AC-Cys18.LC20) was exchanged into turkey gizzard myosin II, it exhibited a 25% fluorescence emission quenching and a 10-nm red-shift upon phosphorylation of serine 19. The myosin II optical biosensor exhibited nearly control levels of the rate of phosphorylation, K+ATPase activity, and in vitro motility. The acrylodan-labeled light chain was exchanged into the A-bands of chicken pectoralis myofibrils in situ to demonstrate the localization and activity of the biosensor in a highly ordered contractile system. Fluorometry and quantitative fluorescence microscopic imaging experiments demonstrated that AC-Cys18.LC20 exchanged myofibrils expressed a phosphorylation-dependent fluorescence change. Labeled light chains were also incorporated into stress fibers of living fibroblasts and smooth muscle cells. This general approach of combining molecular biology and fluorescence spectroscopy to create novel protein-based optical biosensors should provide valuable tools for investigations with model systems and solution studies and ultimately yield important information about temporal-spatial chemical and molecular changes in live cells.

Highly Polarized EGF Receptor Tyrosine Kinase Activity Initiates Egg Activation in Xenopus

Progesterone-matured Xenopus oocytes are arrested at second metaphase but resume meiosis following fertilization. To explore the role of tyrosine kinase activity and phosphatidylinositol turnover in this activation process, we caused oocytes to express three types of human epidermal growth factor receptor (EGF-R), which differ in their ability to stimulate these biochemical processes. Following mRNA injection we found that receptor expression was highly polarized, with most receptors located on the animal hemisphere. Occupancy of the wild-type EGF-R in progesterone-matured oocytes resulted in full egg activation as indicated by an activation potential, increased intracellular-free Ca2+ ([Ca2+]i), fertilization envelope liftoff, and cortical contraction. Fura-2 imaging showed that the wave of EGF-mediated Ca2+ release started in the animal hemisphere and progressed completely around the cell. These responses required receptor tyrosine kinase activity. Matured oocytes expressing the c'973 EGF-R, which possesses kinase activity but only weakly stimulates phosphatidylinositol turnover, responded differently to EGF addition. Cortical contraction and fertilization envelope liftoff appeared normal, but there was no activation potential. Significantly, [Ca2+]i was only slightly elevated and was topologically restricted to the regions expressing receptors. Our results suggest that some aspects of egg activation can occur through a tyrosine kinase pathway. However, phosphatidylinositol hydrolysis appears necessary for both amplification and propagation of signals generated locally by activated EGF-R.

The role of protein kinase C in reorganization of the cortical cytoskeleton during the transition from oocyte to fertilization-competent egg.

Fertilization-competent amphibian eggs (metaphase II) are programmed to undergo an actin-myosin based contraction of the cortical cytoplasm (i.e., cortical contraction) in response to an elevation of intracellular-free calcium which accompanies fertilization. This ability to undergo cortical contraction is acquired within a few hours after the meiotically-arrested oocyte is triggered to resume meiosis by exposure to progesterone. This report examines the timing of changes in the contractile potential of the cortical cytoplasm as the oocyte becomes the egg, and in addition, the signal transduction events which induce these changes. We use the bisected oocyte system developed by Christensen et al. ('84; Nature 310: 150-151) to assess the changes in cortical potential during the meiotic resumption. Immediately after progesterone treatment (less than 5% of the way through the meiotic resumption) the cortex acquires the ability to form a contractile ring, an ability which gradually disappears during the meiotic resumption. Eighty percent of the way through the meiotic resumption the cortex of the hemisphere rapidly acquires the ability to undergo cortical contraction. In contrast, when bisected in a medium containing protein kinase C (PKC) agonists, the cortex of the hemisphere undergoes cortical contraction much earlier (i.e., 50% through the meiotic resumption). In addition, treatment of oocytes with PKC agonists alone can mimic the complete spectrum of changes in cortical potential induced by progesterone, suggesting that PKC has a role in reorganization of the cortical cytoskeleton which occurs as a normal response to progesterone. In support of this, antagonists of PKC block the progesterone-induced reorganization of the cortical cytoskeleton.

Activation of Xenopus laevis eggs in the absence of intracellular Ca activity by the protein phosphorylation inhibitor, 6-dimethylaminopurine (6-DMAP).

Xenopus laevis eggs pricked or microinjected with water or saline in medium containing a limited quantity of free Ca (1.0 to 2.0 microM) remain unactivated for at least 6 hr, even after transfer to oocyte medium containing Ca at higher concentrations (0.5-1.0 mM). These injected eggs, when later pricked in oocyte medium or exposed to A23187 or urethane are fully capable of activation. This confirms the observations of Wangh ('89). However, eggs injected in this Ca-limited medium (CaLM) with 6-DMAP as well as those simply exposed to this drug undergo changes characteristic of activation, including cortical contraction, cortical granule breakdown, a loss of MPF and CSF activities, and pronuclear formation. The time required for 6-DMAP to induce egg activation is inversely correlated to its concentration. Interestingly, eggs that have been injected with EGTA, and thus are unable to respond to activation stimuli such as pricking and A23187 or urethane treatment, can also be activated by exposure to 6-DMAP. In contrast, eggs exposed to or injected with a 6-DMAP analogue (6-aminopurine or puromycin) or a protein synthesis inhibitor (cycloheximide or emetine or puromycin) are not activated. As well, eggs injected in CaLM with 6-DMAP simultaneously with a phosphatase inhibitor (NaF or ammonium molybdate) fail to become activated. Although 6-DMAP-activated eggs remain at the pronucleus stage so long as 6-DMAP is present, they resume cell cycle activities after the drug is withdrawn. They form cleavage furrows, disassemble pronuclear envelopes, and recondense chromosomes. Also, MPF activity reappears and cycles at least twice, peaking each time shortly before cleavage furrow formation. These results suggest that activation of Xenopus eggs arrested at metaphase II by inhibition of protein phosphorylation does not require intracellular Ca release and that maintenance of the egg at metaphase II depends upon continuous protein phosphorylation.

3 Actin and Actin-Associated Proteins in Xenopus Eggs and Early Embryos: Contribution to Cytoarchitecture and Gastrulation

Publisher Summary This chapter discusses the contribution of actin to the events of oogenesis and early embryogenesis in eggs and embryos of the frog X. laevis. This contribution includes the organization of the mitochondrial cloud and the maintenance of the vegetal shell of cytoskeletal components that limits the diffusion of Veg-1 mRNA during oogenesis, the cortical contractions that occur following fertilization, the localization of as yet undefined developmental cues in appropriate places within the cytoplasm and their appropriate apportionment to the daughter cells during cytokinesis, and the cell shape changes and motility that produce gastrulation. In addition, preliminary results have been described in the chapter showing that a group of proteins antigenically related to an actin-associated antigen, the MAb 2E4 antigen, is localized in regions of the egg and early embryo, where actin filaments play an incisive role in these events. Finally, it has been shown that at least one of these proteins, the 48-kDa doublet, recognized by antiserum 3, is preferentially expressed prior to midblastula transition in the subpopulation of the cells that begin the movements of gastrulation— that is, the deep cells of the dorsal lip of the blastopore.

Intracellular pH and intracellular free calcium responses to protein kinase C activators and inhibitors in Xenopus eggs

Cell activation during fertilization of the egg of Xenopus laevis is accompanied by various metabolic changes, including a permanent increase in intracellular pH (pHi) and a transient increase in intracellular free calcium activity ([Ca2+]i). Recently, it has been proposed that protein kinase C (PKC) is an integral component of the Xenopus fertilization pathway (Bement and Capco, J. Cell Biol. 108, 885-892, 1989). Indeed, activators of PKC trigger cortical granule exocytosis and cortical contraction, two events of egg activation, without, however, releasing the cell cycle arrest (blocked in second metaphase of meiosis). In the egg of Xenopus, exocytosis as well as cell cycle reinitiation are supposed to be triggered by the intracellular Ca2+ transient. We report here that PKC activators do not induce the intracellular Ca2+ transient, or the activation-associated increase in pHi. These results suggest that the ionic responses to egg activation in Xenopus do not appear to depend on the activation of PKC. In addition, in eggs already pretreated with phorbol esters, those artificial activators that act by releasing Ca2+ intracellularly, triggered a diminished increase in pHi. Finally, sphingosine and staurosporine, two potent inhibitors of PKC, were found to trigger egg activation, suggesting that a decrease in PKC activity might be an essential event in the release of the metaphase block, in agreement with recent findings on the release of the prophase block in Xenopus oocytes (Varnold and Smith, Development 109, 597-604, 1990).