A cadherin-like protein in eggs and cleaving embryos of Xenopus laevis is expressed in oocytes in response to progesterone

A new cadherin-like protein (CLP) was identified in oocytes, eggs, and cleavage stage embryos of Xenopus laevis. As a probe for detecting new cadherin proteins, an antiserum was raised to a 17 amino acid peptide derived from a highly conserved region in the cytoplasmic domain of all cadherins which have been sequenced to date. This antipeptide antibody recognized Xenopus E-cadherin and a polypeptide in Xenopus brain extracts similar to N-cadherin, which were independently identified by specific mAbs. In extracts of eggs and midblastula stage embryos the antipeptide antibody recognized specifically a 120-kD glycoprotein that migrated faster on SDS gels than the 140-kD E- and N-cadherin polypeptides. This 120-kD polypeptide was not recognized by the mAbs specific to E- and N-cadherin. In fact, E- and N-cadherin were not detectable in eggs or midblastula stage embryos. The possible relationship of CLP to P-cadherin, which has been identified in mouse tissues, has not yet been determined. CLP was synthesized by large, late stage oocytes. When oocytes were induced to mature in vitro with progesterone it accumulated to the same level found in normally laid eggs. It did not accumulate further to any significant extent during the early cleavage stages. CLP was detected on the surface of stage 8 blastomeres by cell surface biotinylation, but only after the tight junctions of the blastula epithelium were opened by removal of Ca2+. We conclude that CLP is a maternally encoded protein that is the major, if not only, cadherin-related protein present in the earliest stages of Xenopus development, and we propose that it may play a role in the Ca2(+)-dependent adhesion and junction formation between cleavage stage blastomeres.     

Cell cycle regulation of pEg3, a new Xenopus protein kinase of the KIN1/PAR-1/MARK family.

We report the characterization of pEg3, a Xenopus protein kinase related to members of the KIN1/PAR-1/MARK family. The founding members of this newly emerging kinase family were shown to be involved in the establishment of cell polarity and both microtubule dynamic and cytoskeleton organization. Sequence analyses suggest that pEg3 and related protein kinases in human, mouse, and Caenorhabditis elegans might constitute a distinct group in this family. pEg3 is encoded by a maternal mRNA, polyadenylated in unfertilized eggs and specifically deadenylated in embryos. In addition to an increase in expression, we have shown that pEg3 is phosphorylated during oocyte maturation. Phosphorylation of pEg3 is cell cycle dependent during Xenopus early embryogenesis and in synchronized cultured XL2 cells. In embryos, the kinase activity of pEg3 is correlated to its phosphorylation state and is maximum during mitosis. Using Xenopus egg extracts we demonstrated that phosphorylation occurs at least in the noncatalytic domain of the kinase, suggesting that this domain might be important for pEg3 function.     

Cell cycle regulation of dynein association with membranes modulates microtubule-based organelle transport

Cytoplasmic dynein is a minus end-directed microtubule motor that performs distinct functions in interphase and mitosis. In interphase, dynein transports organelles along microtubules, whereas in metaphase this motor has been implicated in mitotic spindle formation and orientation as well as chromosome segregation. The manner in which dynein activity is regulated during the cell cycle, however, has not been resolved. In this study, we have examined the mechanism by which organelle transport is controlled by the cell cycle in extracts of Xenopus laevis eggs. Here, we show that photocleavage of the dynein heavy chain dramatically inhibits minus end-directed organelle transport and that purified dynein restores this motility, indicating that dynein is the predominant minus end-directed membrane motor in Xenopus egg extracts. By measuring the amount of dynein associated with isolated membranes, we find that cytoplasmic dynein and its activator dynactin detach from the membrane surface in metaphase extracts. The sevenfold decrease in membrane-associated dynein correlated well with the eightfold reduction in minus end-directed membrane transport observed in metaphase versus interphase extracts. Although dynein heavy or intermediate chain phosphorylation did not change in a cell cycle- dependent manner, the dynein light intermediate chain incorporated approximately 12-fold more radiolabeled phosphate in metaphase than in interphase extracts. These studies suggest that cell cycle-dependent phosphorylation of cytoplasmic dynein may regulate organelle transport by modulating the association of this motor with membranes.     

Four-dimensional imaging of cytoskeletal dynamics in Xenopus oocytes and eggs

The Xenopus laevis (African clawed frog) system has long been popular for studies of both developmental and cell biology, based on a variety of its intrinsic features including the large size of Xenopus oocytes, eggs, and embryos, and the relative ease of manipulation. Unfortunately, the large size has also been considered a serious impediment for high-resolution light microscopy, as has the heavy pigmentation. However, the recent development and exploitation of 4D imaging approaches, and the fact that much of what is of most interest to cell and developmental biologists takes place near the cell surface, indicates that such concerns are no longer valid. Consequently, the Xenopus system in many respects is now as good as other model systems considered to be ideal for microscopy-based studies. Here, 4D imaging and its recent applications to cytoskeletal imaging in Xenopus oocytes and eggs are discussed.     

Chromosome replication in cell-free systems from Xenopus eggs

Cell-free systems from eggs of the frog Xenopus laevis are able to perform most of the acts of eukaryotic chromosome replication in vitro. This now includes the crucial regulatory step of initiation, which had only been achieved for viral systems previously. Purified DNA or nuclei are able to initiate and complete semi-conservation replication in egg extracts in vitro (Blow & Laskey, Cell 47, 557-587 (1986). Replication does not require specialized DNA sequences either in vitro or in microinjected eggs, but in both systems large templates replicate more efficiently than small templates. In some cases replication can re-initiate, excluding the possibility that replication is primed by preexisting primers in the template preparations. When nuclei are replicated in vitro, only one round of replication is observed in a single incubation resembling the single round of replication observed for purified DNA after micro-injection. The mechanism that prevents re-initiation of replication within a single cell cycle is discussed and certain models are eliminated. Nucleosome assembly from histones and DNA has also been studied in cell-free systems from Xenopus eggs. Fractionation has led to the identification of two acidic proteins called nucleoplasmin and N1, which bind histones and transfer them to DNA. The sequences of both proteins have been determined by cDNA cloning and sequencing. Both proteins are found as complexes with histones in eggs.     

Reconstitution of Src-dependent phospholipase Cgamma phosphorylation and transient calcium release by using membrane rafts and cell-free extracts from Xenopus eggs

We reported previously that egg membrane rafts serve as a subcellular microdomain for sperm-dependent tyrosine kinase signaling in Xenopus fertilization. Moreover, we demonstrated that raft-associated Src tyrosine kinase was activated by sperm in vitro. Here we show that egg rafts incubated with sperm or hydrogen peroxide (H2O2) can promote Src-dependent phosphorylation of phospholipase Cgamma (PLCgamma) and transient calcium release in the extracts of unfertilized Xenopus eggs. In vivo egg activation by sperm or H2O2 also promotes tyrosine phosphorylation and raft-translocalization of PLCgamma. Immunodepletion of PLCgamma from the egg extracts inhibits the raft-dependent calcium release. Rafts prepared from H2O2-activated eggs also promote Src-dependent dephosphorylation of p42 mitogen-activated protein kinase and cell cycle transition from metaphase II to interphase in egg extracts. PLCgamma phosphorylation and calcium release in egg extracts can be promoted by rafts prepared from COS-7 cells expressing the Xenopus Src gene. These results demonstrate that the signaling events elicited by fertilization in Xenopus eggs can be reconstituted in vitro. The development of such experimental platforms will allow us to dissect the molecular mechanism of sperm-dependent activation of raft-associated Src and subsequent up-regulation of PLCgamma and egg activation machinery in Xenopus eggs.     

Fission yeast p13 blocks mitotic activation and tyrosine dephosphorylation of the Xenopus cdc2 protein kinase

It has been demonstrated that the Xenopus homolog of the fission yeast cdc2 protein is a component of M phase promoting factor (MPF). We show that the Xenopus cdc2 protein is phosphorylated on tyrosine in vivo, and that this tyrosine phosphorylation varies markedly with the stage of the cell cycle. Tyrosine phosphorylation is high during interphase (in Xenopus oocytes and activated eggs) but absent during M phase (in unfertilized eggs). In vitro activation of pre-MPF from Xenopus oocytes results in tyrosine dephosphorylation of the cdc2 protein and switching-on of its kinase activity. The product of the fission yeast suc1 gene (p13), which inhibits the entry into mitosis in Xenopus extracts, completely blocks tyrosine dephosphorylation and kinase activation. However, p13 has no effect on the activated form of the cdc2 kinase. These findings suggest that p13 controls the activation of the cdc2 kinase, and that tyrosine dephosphorylation is an important step in this process.     

A member of the Ste20/PAK family of protein kinases is involved in both arrest of Xenopus oocytes at G2/prophase of the first meiotic cell cycle and in prevention of apoptosis.

We have identified new members (X-PAKs) of the Ste20/PAK family of protein kinases in Xenopus, and investigated their role in the process that maintains oocytes arrested in the cell cycle. Microinjection of a catalytically inactive mutant of X-PAK1 with a K/R substitution in the ATP binding site, also deleted of its Nter-half that contains the conserved domains responsible for binding of both Cdc42/Rac GTPases and SH3-containing proteins, greatly facilitates oocyte release from G2/prophase arrest by progesterone and insulin. Addition of the same X-PAK1 mutant to cell cycle extracts from unfertilized eggs induced apoptosis, as shown by activation of caspases and cytological changes in in vitro-assembled nuclei. This was suppressed by adding Bcl-2 or the DEVD peptide inhibitor of caspases, and rescued by competing the dominant-negative mutant with its constitutively active X-PAK1 counterpart. Such results indicate that X-PAK1 (or another member of the Xenopus Ste20/PAK family of protein kinases) is involved in arrest of oocytes at G2/prophase and prevention of apoptosis; thus death by apoptosis and release of healthy oocytes from cell cycle arrest may be linked. That cell cycle arrest protects oocytes from apoptosis is consistent with the finding that extracts from metaphase II-arrested oocytes are less sensitive to apoptotic signals than those from activated eggs.     

A highly conserved Poc1 protein characterized in embryos of the hydrozoan Clytia hemisphaerica: localization and functional studies

Poc1 (Protein of Centriole 1) proteins are highly conserved WD40 domain-containing centriole components, well characterized in the alga Chlamydomonas, the ciliated protazoan Tetrahymena, the insect Drosophila and in vertebrate cells including Xenopus and zebrafish embryos. Functions and localizations related to the centriole and ciliary axoneme have been demonstrated for Poc1 in a range of species. The vertebrate Poc1 protein has also been reported to show an additional association with mitochondria, including enrichment in the specialized "germ plasm" region of Xenopus oocytes. We have identified and characterized a highly conserved Poc1 protein in the cnidarian Clytia hemisphaerica. Clytia Poc1 mRNA was found to be strongly expressed in eggs and early embryos, showing a punctate perinuclear localization in young oocytes. Fluorescence-tagged Poc1 proteins expressed in developing embryos showed strong localization to centrioles, including basal bodies. Anti-human Poc1 antibodies decorated mitochondria in Clytia, as reported in human cells, but failed to recognise endogenous or fluorescent-tagged Clytia Poc1. Injection of specific morpholino oligonucleotides into Clytia eggs prior to fertilization to repress Poc1 mRNA translation interfered with cell division from the blastula stage, likely corresponding to when neosynthesis normally takes over from maternally supplied protein. Cell cycle lengthening and arrest were observed, phenotypes consistent with an impaired centriolar biogenesis or function. The specificity of the defects could be demonstrated by injection of synthetic Poc1 mRNA, which restored normal development. We conclude that in Clytia embryos, Poc1 has an essentially centriolar localization and function.     

Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins

The use of cell-free extracts prepared from eggs of the South African clawed toad, Xenopus laevis, has led to many important discoveries in cell cycle research. These egg extracts recapitulate the key nuclear transitions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. DNA added to the extract is first assembled into a nucleus and is then efficiently replicated. Progression of the extract into mitosis then allows the separation of paired sister chromatids. The Xenopus cell-free system is therefore uniquely suited to the study of the mechanisms, dynamics and integration of cell cycle regulated processes at a biochemical level. In this article we describe methods currently in use in our laboratory for the preparation of Xenopus egg extracts and demembranated sperm nuclei for the study of DNA replication in vitro. We also detail how DNA replication can be quantified in this system. In addition, we describe methods for isolating chromatin and chromatin-bound protein complexes from egg extracts. These recently developed and revised techniques provide a practical starting point for investigating the function of proteins involved in DNA replication.     

Changes in intracellular pH following egg activation and during the early cell cycle of the amphibian Pleurodeles waltlii, coincide with changes in MPF activity.

Previous work on Xenopus laevis suggests a temporal coincidence between inactivation of the M-phase promoting factor (MPF) and intracellular pH (pHi) increase during egg activation. In addition, we recently showed that during the early cell cycle of Xenopus eggs, MPF activity cycling and pHi oscillations were temporally and functionally related. In the present work, using eggs of another amphibian, Pleurodeles waltlii, which has a natural cell cycle considerably longer than that of Xenopus laevis, we show a temporal coincidence between MPF activity and pHi changes, both at the time of egg activation and at each of the following cell cycles. Egg activation-induced pHi changes in Pleurodeles did not involve classical plasma membrane ion exchangers, and were not due to the activation of a H+ conductance. On the other hand, the pHi oscillations intervening at each cell cycle were suppressed by inhibitors of protein synthesis or phosphorylation, as were their counterparts in Xenopus eggs. We propose that physiological pHi changes in Pleurodeles and Xenopus eggs might have a metabolic origin, in direct relation with the cascade of phosphorylations-dephosphorylations of proteins implicated in the control of the cell cycle.     

Membrane-associated lamins in Xenopus egg extracts: identification of two vesicle populations

Nuclear lamin isoforms of vertebrates can be divided into two major classes. The B-type lamins are membrane associated throughout the cell cycle, whereas A-type lamins are recovered from mitotic cell homogenates in membrane-free fractions. A feature of oogenesis in birds and mammals is the nearly exclusive presence of B-type lamins in oocyte nuclear envelopes. In contrast, oocytes and early cleavage embryos of the amphibian Xenopus laevis are believed to contain a single lamin isoform, lamin LIII, which after nuclear envelope breakdown during meiotic maturation is reported to be completely soluble. Consequently, we have reexamined the lamin complement of Xenopus oocyte nuclear envelopes, egg extracts, and early embryos. An mAb (X223) specific for the homologous B-type lamins B2 of mouse and LII of Xenopus somatic cells (Hoger, T., K. Zatloukal, I. Waizenegger, and G. Krohne. 1990. Chromosoma. 99:379-390) recognized a Xenopus oocyte nuclear envelope protein biochemically distinct from lamin LIII and very similar or identical to somatic cell lamin LII. Oocyte lamin LII was detectable in nuclear envelopes of early cleavage embryos. Immunoblotting of fractionated egg extracts revealed that approximately 20-23% of lamin LII and 5-7% of lamin LIII were membrane associated. EM immunolocalization demonstrated that membrane-bound lamins LII and LIII are associated with separate vesicle populations. These findings are relevant to the interpretation of nuclear reconstitution experiments using Xenopus egg extracts.     

Dissection of the XChk1 signaling pathway in Xenopus laevis embryos

Checkpoint pathways inhibit cyclin-dependent kinases (Cdks) to arrest cell cycles when DNA is damaged or unreplicated. Early embryonic cell cycles of Xenopus laevis lack these checkpoints. Completion of 12 divisions marks the midblastula transition (MBT), when the cell cycle lengthens, acquiring gap phases and checkpoints of a somatic cell cycle. Although Xenopus embryos lack checkpoints prior to the MBT, checkpoints are observed in cell-free egg extracts supplemented with sperm nuclei. These checkpoints depend upon the Xenopus Chk1 (XChk1)-signaling pathway. To understand why Xenopus embryos lack checkpoints, xchk1 was cloned, and its expression was examined and manipulated in Xenopus embryos. Although XChk1 mRNA is degraded at the MBT, XChk1 protein persists throughout development, including pre-MBT cell cycles that lack checkpoints. However, when DNA replication is blocked, XChk1 is activated only after stage 7, two cell cycles prior to the MBT. Likewise, DNA damage activates XChk1 only after the MBT. Furthermore, overexpression of XChk1 in Xenopus embryos creates a checkpoint in which cell division arrests, and both Cdc2 and Cdk2 are phosphorylated on tyrosine 15 and inhibited in catalytic activity. These data indicate that XChk1 signaling is intact but blocked upstream of XChk1 until the MBT.     

Organisation of Xenopus egg cytoplasm: response to simulated microgravity

The cytoplasm of Xenopus fertilised eggs appears to be organised into three major compartments based primarily on the uneven distribution of yolk platelets. There is a shift of these yolk compartments during the first cell cycle that is thought to be involved in the dorsal/ventral morphogenesis of the embryo. The involvement of gravity in Xenopus cytoplasmic organisation and in compartment shifts was addressed by examining, cytologically, the yolk compartments in embryos that developed under the simulated microgravity conditions of the horizontal clinostat. The cytoplasmic organisation into yolk compartments was found to be maintained, and the asymmetric movements of compartments still occurred in eggs that developed on the clinostat. It is suggested that the organisation of Xenopus egg cytoplasm into discrete compartments relies on forces other than those involving gravity (i.e., not density differences), and that the compartment shifts that take place during the first cell cycle are active movements. The variation in compartment size and composition observed from batch to batch of eggs, and to a lesser extent from egg to egg, during this study was addressed.     

Two-dimensional protein maps of xenopus eggs and embryos at different developmental stages.

Protein expression during the early development of Xenopus has been followed by 2D-polyacrylamide gel electrophoresis (PAGE). The analysis of two-dimensional maps of eggs and embryos at different stages of development has allowed the separation of more than 2000 spots. Identification of numerous polypeptides was obtained in four different ways: (1) immuno-blotting; (2) amino terminal sequence after blotting on to PVDF membranes; (3) comigration; and (4) assignment in comparison with proteins separated by 2D techniques on reference maps such as human liver, red blood cells, plasma and cerebrospinal fluid reported in the Swiss 2D-PAGE Data Base. The maps presented in this report are a step toward the study of the protein expression in Xenopus eggs and embryos and may be a powerful working tool since Xenopus embryos are popular models for the study of development.     

Assembly of nucleosomes: the reaction involving X. laevis nucleoplasmin

We analyze the nucleosome core assembly reaction which is mediated in vitro by a protein previously purified from Xenopus laevis eggs, now named nucleoplasmin in reference to its occurrence in the soluble phase of the nucleus of a wide range of vertebrate cell types. Nucleoplasmin is present in solution as a pentamer. We use nuclease digestion analysis to show that the protein assembles bona fide nucleosome cores in vitro from purified histones and DNA. Nucleoplasmin itself binds neither to DNA nor to the nucleoprotein particles which it assembles in vitro. However, it interacts with histones in vitro in such a way that histones no longer adhere to negatively charged surfaces. We have found no evidence for sterically specific interactions with particular histones. The initial rate of the nucleosome core assembly reaction mediated by purified nucleoplasmin in vitro is essentially identical with the rate of the nucleosome assembly reaction which occurs in the cell-free extracts of Xenopus eggs from which nucleoplasmin was purified. This rate is sufficient to account for the rate of nucleosome assembly required during the early development of Xenopus embryos.     

Multiple forms of DNA-dependent DNA polymerase during early development and in somatic cells of Xenopus laevis.

Four distinct DNA-dependent DNA polymerase activities (DNA polymerases I, II, III and IV according to the order of elution from a DEAE column) have been separated from extracts of unfertilized Xenopus laevis eggs. The same activities, on the basis of their chromatographic properties, template specificities and sedimentation coefficients, have been found in embryos at least until the gastrula stage. On the other hand, Xenopus kidney cells grown in culture, as well as full grown oocytes lack DNA polymerase I. These data suggest the DNA polymerase I might be a special DNA polymerase activity involved in the extremely rapid DNA synthesis which takes place during early development of X. laevis.     

Vimentin expression in oocytes, eggs and early embryos of Xenopus laevis

Immunocytochemical studies using a monoclonal anti-porcine vimentin antibody reveal a well-organized pattern of staining in Xenopus laevis oocytes, eggs and early embryos. The positions of Xenopus vimentin and desmin in two-dimensional (2D) polyacrylamide gels were first established by immunoblotting of muscle Triton extracts with anti-intermediate filament antibodies (anti-IFA), which cross-react with all intermediate filament proteins (IFPs). The anti-porcine vimentin reacts with vimentin and desmin in muscle 2D immunoblots, but only reacts with one polypeptide in oocyte blots in the position predicted for vimentin (Mr 55 x 10(3), pI 5.6). Using an anti-sense probe derived from a Xenopus vimentin genomic clone in RNase protection assays, we show that expression of vimentin begins in previtellogenic oocytes. The level of expression remains constant throughout oogenesis and in unfertilized eggs. These data suggest that vimentin is expressed in oocytes and eggs. Most interestingly, the immunocytochemical results also show that vimentin is present in the germ plasma of oocytes, eggs and early embryos. It is therefore possible that vimentin has an important role in the formation or behaviour of early germ line cells.