Overexpressed Monomeric Human Acetylcholinesterase Induces Subtle Ultrastructural Modifications in Developing Neuromuscular Junctions of Xenopus laevis Embryos

Abstract: Formation of a functional neuromuscular junction (NMJ) involves the biosynthesis and transport of numerous muscle-specific proteins, among them the acetylcholine-hydrolyzing enzyme acetylcholinesterase (AChE). To study the mechanisms underlying this process, we have expressed DMA encoding human AChE downstream of the cytomegalovirus promoter in oocytes and developing embryos of Xenopus laevis. Recombinant human AChE (rHAChE) produced in Xenopus was biochemically and immunochemically indistinguishable from native human AChE but clearly distinguished from the endogenous frog enzyme. In microinjected embryos, high levels of catalytically active rHAChE induced a transient state of over-expression that persisted for at least 4 days postfertilization. rHAChE appeared exclusively as nonassembled monomers in embryos at times when endogenous Xenopus AChE displayed complex oligomeric assembly. Nonetheless, cell-associated rHAChE accumulated in myotomes of 2-and 3-day-old embryos within the same sub-cellular compartments as native Xenopus AChE. NMJs from 3-day-old DNA-injected embryos displayed fourfold or greater overexpression of AChE, a 30% increase in postsynaptic membrane length, and increased folding of the postsynaptic membrane. These findings indicate that an evolutionarily conserved property directs the intracellular trafficking and synaptic targeting of AChE in muscle and support a role for AChE in vertebrate synaptogenesis.     

Calmodulin triggers the resumption of meiosis in amphibian oocytes

The calcium-binding protein, calmodulin, has been purified from Xenopus laevis oocytes. This 18,500-dalton protein, pl 4.3, has two high-affinity calcium-binding sites per mole protein having a dissociation constant of 2.8 x 10(-6) M. Full-grown Xenopus oocytes, arrested in late G2 of the meiotic cell cycle, resumed meiosis when microinjected with 60-80 ng (3-4 pmol) of calmodulin in the form of a calcium-calmodulin complex. The timing of the meiotic events in these recipient oocytes was the same as that normally induced by progesterone. Xenopus ovarian calmodulin stimulated bovine brain phosphodiesterase (PDE) 3- to 10-fold in a calcium-dependent manner, but it had no apparent effect on ovarian PDE activity. A calcium-calmodulin-dependent protein kinase has been isolated from Xenopus oocytes using a calmodulin-Sepharose 4B affinity column. The possible role for this kinase in regulating the G2-M transition in oocytes has been discussed.     

Early steroid metabolism in Xenopus laevis, Rana temporaria and Triturus vulgaris embryos.

Embryos of Xenopus laevis, Rana temporaria and Triturus vulgaris exposed to radioactive pregnenolone have been found to convert it to progesterone. Incubations with radioactive progesterone showed that it was actively metabolized by oocytes and embryos. In Xenopus incubations progesterone was converted to 5alpha-pregnane-3,20-dione, 17alpha-hydroxy-4-pregnen-3-one, 4-androstene-3,17-dione and 17alpha-20alpha-dihydroxy-4-pregne-3-one, indicating 5alpha-reductase, 17alpha-hydroxylase, 19-20-desmolase and 20alpha-hydroxylase activities. In oocytes of Triturus and Rana no evidence of 19-20-desmolase was found. In Rana oocytes were also not evidence of 17alpha-hydroxylase activity. All identified activities except 20alpha-hydroxylase were common to embryos of all three species. It is suggested that the steroid enzyme activities present in the embryos are not solely derived from the oocytes but synthentized during early development. Possible meaning of this kind of metabolism during differentiation remains open.     

Acetylcholinesterase clustering at the neuromuscular junction involves perlecan and dystroglycan.

Formation of the synaptic basal lamina at vertebrate neuromuscular junction involves the accumulation of numerous specialized extracellular matrix molecules including a specific form of acetylcholinesterase (AChE), the collagenic-tailed form. The mechanisms responsible for its localization at sites of nerve- muscle contact are not well understood. To understand synaptic AChE localization, we synthesized a fluorescent conjugate of fasciculin 2, a snake alpha-neurotoxin that tightly binds to the catalytic subunit. Prelabeling AChE on the surface of Xenopus muscle cells revealed that preexisting AChE molecules could be recruited to form clusters that colocalize with acetylcholine receptors at sites of nerve-muscle contact. Likewise, purified avian AChE with collagen-like tail, when transplanted to Xenopus muscle cells before the addition of nerves, also accumulated at sites of nerve-muscle contact. Using exogenous avian AChE as a marker, we show that the collagenic-tailed form of the enzyme binds to the heparan-sulfate proteoglycan perlecan, which in turn binds to the dystroglycan complex through alpha-dystroglycan. Therefore, the dystroglycan-perlecan complex serves as a cell surface acceptor for AChE, enabling it to be clustered at the synapse by lateral migration within the plane of the membrane. A similar mechanism may underlie the initial formation of all specialized basal lamina interposed between other cell types.     

Early Steroid Metabolism in Xenopus laevis, Rana temporaria and Triturus vulgaris Embryos

Embryos of Xenopus laevis , Rana temporaria and Triturus vulgaris exposed to radioactive pregnenolone have been found to convert it to progesterone. Incubations with radioactive progesterone showed that it was actively metabolized by oocytes and embryos.
In Xenopus incubations progesterone was converted to 5α-pregnane-3,20-dione, 17α-hydroxy-4pregnen-3-one, 4-androstene-3,17-dione and 17α,20α:-dihydroxy-4-pregnen-3-one, indicating 5α-reductase, 17α-hydroxylase, 19–20-desmolase and 20α-hydroxylase activities. In oocytes of Triturus and Rana no evidence of 19–20-desmolase was found. In Rana oocytes were also not evidence of 17α-hydroxylase activity. All identified activities except 20α-hydroxylase were common to embryos of all three species.
It is suggested that the steroid enzyme activities present in the embryos are not solely derived from the oocytes but synthetized during early development. Possible meaning of this kind of metabolism during differentiation remains open.     

Acetylcholinesterase from the Skeletal Muscle of the Lamprey Petromyzon marinus Exists in Globular and Asymmetric Forms

To obtain information about the evolution of acetylcholinesterase (AChE), we undertook a study of the enzyme from the skeletal muscle of the lamprey Petromyzon marinus, a primitive vertebrate. We found that the cholinesterase activity of lamprey muscle is due to AChE, not pseudocholinesterase; the enzyme was inhibited by 1,5-bis(4-allyldimethylammonium phenyl) pentane-3-one (BW284C51), but not by tetramonoisopropyl pyrophosphortetramide (iso-OMPA) or ethopropazine. Also, the enzyme had a high affinity for acetylthiocholine and was inhibited by high concentrations of substrate. A large fraction of the AChE was found to be glycoprotein, since it was precipitated by concanavalin A-agarose. Optimal extraction of AChE was obtained in a high-salt detergent-containing buffer; fractional amounts of enzyme were extracted in buffers lacking salt and/or detergent. These data suggest that globular and asymmetric forms of AChE are present. On sucrose gradients, enzyme that was extracted in high-salt detergent-containing buffer sedimented as a broad peak of activity corresponding to G4; additionally, there was usually a peak corresponding to A12. Sequential extraction of AChE in conjunction with velocity sedimentation resolved minor forms of AChE and revealed that the G1, G2, G4, A4, A8, and A12 forms of AChE could be obtained from the muscle. The identity of the forms was confirmed through high-salt precipitation and collagenase digestion. The asymmetric forms of AChE were precipitated in low ionic strength buffer, and their sedimentation coefficients were shifted to higher values by collagenase digestion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunological identification of the karyophilic, histone-binding proteins N1 and N2 in somatic cells and oocytes of diverse amphibia

A polypeptide pair designated N1/N2 (Mr 100 000 and 110 000) is an exceptionally acidic and abundant nuclear protein of oocytes of the toad, Xenopus laevis, and is characterized by a pronounced karyophilia. These proteins have been shown to form specific complexes with free, i.e., non-chromatin-bound histones H3 and H4 (Kleinschmidt & Franke, Cell 29 (1982) 799) [3]. In order to study these proteins and their possible counterparts in other species, antibodies were produced in guinea pigs against proteins N1/N2 purified from Xenopus oocyte nuclei. Using gel electrophoresis, peptide map analysis, immunoblotting techniques and immuno fluorescence microscopy the existence of polypeptides identical in Mr value and charge to polypeptide N1 of oocytes was demonstrated in cultured somatic cells of Xenopus laevis, where it was also highly enriched in cell nuclei, although the cellular concentration was much lower than in oocytes. A similar, if not identical protein, was recognized in nuclei of diverse other cell types including hepatocytes, enterocytes, ovarian follicle cells, and Sertoli cells of testis, of Xenopus, Rana temporaria, R. esculenta, Pleurodeles waltlii but not in erythrocytes and later stages of spermiogenesis. When nuclear proteins from oocytes of different amphibian species were examined with these antibodies it was found that the Mr values of N1/N2 proteins were considerably different in different species, ranging from Mr 110 000 to 190 000. Immunoprecipitation and gel electrophoretic analysis under non-denaturing conditions showed that a significant proportion of these proteins was contained in complexes with histones H3 and H4. The results demonstrate that proteins N1/N2 are not special proteins of oocytes of Xenopus laevis but occur in various other cells of diverse amphibian species. The widespread occurrence of these karyophilic proteins indicates that at least one function of these proteins, i.e., selective binding of the arginine-rich histones H3 and H4, is not exclusive to oocytes but may also contribute to the regulation of histone pools and chromatin formation in other cell types.     

ATP crossing the cell plasma membrane generates an ionic current in xenopus oocytes

The presence of ATP within cells is well established. However, ATP also operates as an intercellular signal via specific purinoceptors. Furthermore, nonsecretory cells can release ATP under certain experimental conditions. To measure ATP release and membrane currents from a single cell simultaneously, we used Xenopus oocytes. We simultaneously recorded membrane currents and luminescence. Here, we show that ATP release can be triggered in Xenopus oocytes by hyperpolarizing pulses. ATP release (3.2 +/- 0.3 pmol/oocyte) generated a slow inward current (2.3 +/- 0.1 microA). During hyperpolarizing pulses, the permeability for ATP(4-) was more than 4000 times higher than that for Cl(-). The sensitivity to GdCl(3) (0. 2 mm) of hyperpolarization-induced ionic current, ATP release and E-ATPase activity suggests their dependence on stretch-activated ion channels. The pharmacological profile of the current inhibition coincides with the inhibition of ecto-ATPase activity. This enzyme is highly conserved among species, and in humans, it has been cloned and characterized as CD39. The translation, in Xenopus oocytes, of human CD39 mRNA encoding enhances the ATP-supported current, indicating that CD39 is directly or indirectly responsible for the electrodiffusion of ATP.     

Teleost Fh14-3-3a protein protects Xenopus oocytes from hyperosmolality

We have previously cloned and characterized a novel 14-3-3 gene from the euryhaline telost Fundulus heteroclitus, Fh14-3-3a (Kültz et al., 2001). The corresponding gene product is osmoregulated and most highly expressed in gill epithelium of this fish. In the present study we have expressed Fh14-3-3a cRNA in Xenopus laevis oocytes and investigated the survival and electrophysiological parameters of Xenopus oocytes in isosmotic and various hyperosmotic media. Xenopus oocytes expressing Fh14-3-3a show no mortality after a 16 hour exposure to hyperosmolality in the form of elevating medium K(+), Na(+), polyethylene glycol, or sorbitol concentrations up to 444 mosmol/kg. In contrast, 16 hours of the same hyperosmolality caused 100% mortality in control Xenopus oocytes injected with water. As a result of hyperosmolality the Xenopus oocyte membrane potential decreased between 10 and 70% in oocytes expressing Fh14-3-3a whereas it was completely abolished in control oocytes. We report that one potential cause for the osmoprotective effect of Fh14-3-3a on Xenopus oocytes could be its inhibition of an endogenous chloride current. Hyperosmotic urea was not as harmful to Xenopus oocytes as hypertonicity and maybe acting through a different mechanism. Coexpression of Fh14-3-3a with a human calcium channel in Xenopus oocytes did not affect the electrophysiological properties of this exogenous channel. Thus, the osmoprotective effect of Fh14-3-3a may prove a valuable tool for the characterization of exogenous ion channels in Xenopus oocytes exposed to hyperosmotic conditions.     

Interaction of eIF4G with poly(A)-binding protein stimulates translation and is critical for Xenopus oocyte maturation

The poly(A)-binding protein Pab1p interacts directly with the eukaryotic translation initiation factor 4G (eIF4G) to facilitate translation initiation of polyadenylated mRNAs in yeast [1,2]. Although the eIF4G-PABP interaction has also been demonstrated in a mammalian system [3,4], its biological significance in vertebrates is unknown. In Xenopus oocytes, cytoplasmic polyadenylation of several mRNAs coincides with their translational activation and is critical for maturation [5-7]. Because the amount of PABP is very low in oocytes [8], it has been argued that the eIF4G-PABP interaction does not play a major role in translational activation during oocyte maturation. Also, overexpression of PABP in Xenopus oocytes has only a modest stimulatory effect on translation of polyadenylated mRNA and does not alter either the efficiency or the kinetics of progesterone-induced maturation [9]. Here, we report that the expression of an eIF4GI mutant defective in PABP binding in Xenopus oocytes reduces translation of polyadenylated mRNA and dramatically inhibits progesterone-induced maturation. Our results show that the eIF4G-PABP interaction is critical for translational control of maternal mRNAs during Xenopus development.     

Enzymatic conversion of adenosine to inosine in the wobble position of yeast tRNAAsp: the dependence on the anticodon sequence.

We have investigated the specificity of the tRNA modifying enzyme that transforms the adenosine at position 34 (wobble position) into inosine in the anticodon of several tRNAs. For this purpose, we have constructed sixteen recombinants of yeast tRNAAsp harboring an AXY anticodon (where X or Y was one of the four nucleotides A, G, C or U). This was done by enzymatic manipulations in vitro of the yeast tRNAAsp, involving specific hydrolysis with S1-nuclease and RNAase A, phosphorylation with T4-polynucleotide kinase and ligation with T4-RNA ligase: it allowed us to replace the normal anticodon GUC by trinucleotides AXY and to introduce simultaneously a 32P-labelled phosphate group between the uridine at position 33 and the newly inserted adenosine at position 34. Each of these 32P-labelled AXY "anticodon-substituted" yeast tRNAAsp were microinjected into the cytoplasm of Xenopus laevis oocytes and assayed for their capacity to act as substrates for the A34 to I34 transforming enzyme. Our results indicate that: 1/ A34 in yeast tRNAAsp harboring the arginine anticodon ACG or an AXY anticodon with a purine at position 35 but with A, G or C but not U at position 36 were efficiently modified into I34; 2/ all yeast tRNAAsp harboring an AXY anticodon with a pyrimidine at position 35 (except ACG) or uridine at position 36 were not modified at all. This demonstrates a strong dependence on the anticodon sequence for the A34 to I34 transformation in yeast tRNAAsp by the putative cytoplasmic adenosine deaminase of Xenopus laevis oocytes.     

Ribonucleotide reductase activity during amphibian development.

Uniformly labeled [3H] uridine is incorporated into DNA by dissociated Pleurodeles blastulae; the label is found in cytosine and to a much lesser extent in thymine. Ribonucleotide reductase activity cannot be detected in full grown oocytes of Xenopus and Pleurodeles, but is present in unfertilized egg. The enzyme is synthesized (or activated) when maturation is induced in Xenopus oocytes by in vitro hormonal treatment. The enzymatic activity increases after fertilization and reaches a peak at the 2--4 cell stage; it decreases at the blastula, gastrula and neurula stages to the low level initially present in unfertilized eggs. The enzyme is no longer detectable in swimming tadpoles. Addition of hydroxyurea (1 mg/ml) to fertilized eggs leads to complete loss of ribonucleotide reductase activity: cycloheximide (20 mug/ml) inhibits the rise in activity characteristic of early cleavage, while actinomycin D (20 mug/ml) has no effect. The significance of these results in discussed.     

Characteristics of the Na+/K+-ATPase from Torpedo californica expressed in Xenopus oocytes: a combination of tracer flux measurements with electrophysiological measurements

The Na+/K+-ATPase from electroplax of Torpedo californica was incorporated into the plasma membrane of Xenopus oocytes by microinjection of mRNA coding for the alpha- and beta-subunit of the enzyme; the mRNAs were obtained by in vitro translation of cloned cDNAs (Noguchi et al. (1988) FEBS Lett. 225, 27-32). (1) Measurements of ouabain-sensitive membrane current revealed that the Na+/K+-ATPase of Torpedo is less sensitive to ouabain than the endogenous enzyme. (2) The ouabain-sensitive membrane currents in mRNA-injected oocytes exhibit similar voltage dependence as the currents generated by the endogenous ATPase of Xenopus oocytes; in particular, the current-voltage relation exhibits a maximum and a negative slope at potentials more positive than +20 mV. (3) A maximum can also be detected if the rate of 22Na+ efflux is determined under different voltage-clamp conditions. If membrane current and rate of Na+2 efflux are determined simultaneously, a voltage-independent ratio between current and flux is obtained suggesting voltage-independent Na+-K+ stoichiometry. The data are compatible with a 3Na+-2K+ stoichiometry.     

Post-translational processing in Xenopus oocytes includes carboxyl-terminal amidation

Xenopus oocytes are versatile cells capable of carrying out many post-translational processes. Although previously reported not to be capable of C-terminal amidation, this report demonstrates that Xenopus oocytes do indeed have an amidating enzyme. The amidating activity from Xenopus ovaries is compared to the known amidating activity found in porcine pituitaries. The demonstration of C-terminal amidation by Xenopus oocytes extends their usefulness in studying post-translational events.     

Purification and characterization of a casein-kinase-II-type enzyme from Xenopus laevis ovary. Biological effects on the meiotic cell division of full-grown oocyte

A casein kinase of type II has been highly purified from Xenopus laevis ovary. A new experimental protocol has been developed for the purification, consisting in four chromatographic steps: hydrophobic on tyrosine-agarose, ion exchange on DEAE-Sepharose, affinity on heparin-Sepharose and fast protein liquid on Mono Q. The purification was greater than 20,000, taking into account an inhibitor present in the starting material which masked the activity in the crude fraction. The overall yield was greater than 20%. Full-grown Xenopus oocytes contain 64 milliunits per oocyte corresponding to an intracellular concentration in the nanomolar range. The enzyme shares the following features with the mammalian casein kinase II: (a) comparable subunit composition (42-kDa doublet, 38 kDa and 26 kDa), (b) autophosphorylation of the 26-kDa subunit, (c) ability to use GTP as well as ATP as phosphate donor, (d) inability to use Mn2+ instead of Mg2+ to support the activity, (e) phosphorylation of both threonine and serine residues of casein, (f) inhibition by low doses of heparin. Biological effects of the highly purified enzyme have been investigated upon microinjection into Xenopus full-grown oocytes. At nanomolar concentrations (approximately 3 nM) the enzyme inhibited progesterone induction of meiotic cell division whereas it facilitates meiotic maturation at the level of maturation-promoting factor. These results suggest a role for the kinase in the phosphorylation cascade involved during the prophase/metaphase transition of meiotic cell division, both in the mechanism of the meiotic prophase arrest and in the activity of the cytoplasmic factor maturation-promoting factor. When microinjected into oocytes above 45 nM, the kinase provoked complex changes in the profile of the in ovo 32P-labelled proteins indicating that its targets could be other kinase/phosphatase regulatory proteins.     

Effect of fibrillation on acetylcholinesterase mRNA in cultured embryonic rat myotubes

Acetylcholinesterase (AChE) and AChE mRNA were evaluated in spontaneously fibrillating myotubes derived from 20-day-old rat fetuses and in matched cultures in which fibrillation was prevented by adding tetrodotoxin on the fourth day of culture. On the eighth day of culture, the AChE activity of fibrillating and nonfibrillating cultures was 5332 and 1861 pmol ACh hydrolyzed min-1 dish-1, respectively (P less than 0.005). Total mRNA was essentially the same in fibrillating and nonfibrillating cultures (27.4 and 25.4 micrograms/dish, respectively). AChE mRNA was assessed by assaying the AChE produced by Xenopus oocytes microinjected with purified mRNA. The AChE produced by mRNA from fibrillating and nonfibrillating cultures was 0.46 and 0.10 pmol ACh hydrolyzed min-1 oocyte-1, respectively (P less than 0.005).     

Cloning and characterization of the gene for the somatic form of DNA topoisomerase I from Xenopus laevis.

Two distinct tissue-specific forms of DNA topoisomerase I with M(r) of 165 and 110 kDa have been purified from oocytes and somatic cells respectively of the African frog Xenopus laevis. In this paper, cDNAs encoding a Xenopus topoisomerase I were cloned using PCR primers derived from sequences of yeast and human topoisomerase I. A polypeptide expressed from a portion of the coding sequence was recognized by an antiserum directed against the somatic topoisomerase I that had previously been shown to be unable to cross-react with the oocyte enzyme. Thus, the clone encodes the somatic cell topoisomerase I. An antiserum raised against a synthetic peptide containing the sequence surrounding the active site tyrosine of the somatic topoisomerase I reacts with the enzymes purified from both oocytes and somatic cells, indicating that the two enzymes share some limited sequence homology. RNA blot hybridization showed that oocytes contain an abundant store of somatic topoisomerase I mRNA that is not efficiently polyadenylated in oocytes. This stored RNA contains a consensus cytoplasmic polyadenylation element that is found in a variety of mRNAs that are translationally repressed in oocytes. Microinjection into oocytes of in vitro transcribed mRNA prepared from a Myc-tagged construct of the somatic topoisomerase I sequence is translated to yield a 110 kDa product. This suggests that the oocyte-specific 165 kDa topoisomerase I is not produced by tissue-specific post-translational modification of the somatic topoisomerase I. The oocyte enzyme appears to be produced from a minor mRNA species in oocytes that has not yet been identified.     

Phosphorylation of Na,K-ATPase alpha-subunits in microsomes and in homogenates of Xenopus oocytes resulting from the stimulation of protein kinase A and protein kinase C.

The phosphorylation of the alpha-subunit of Na+/K(+)-transporting ATPase (Na,K-ATPase) by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was characterized in purified enzyme preparations of Bufo marinus kidney and duck salt gland and in microsomes of Xenopus oocytes. In addition, we have examined cAMP and phorbol esters, which are stimulators of PKA and PKC, respectively, for their ability to provoke the phosphorylation of alpha-subunits of Na,K-ATPase in homogenates of Xenopus oocytes. In the enzyme from the duct salt gland, phosphorylation by PKA and PKC occurs on serine and threonine residues, whereas in the enzyme from B. marinus kidney and Xenopus oocytes, phosphorylation by PKA occurs only on serine residues. Phosphopeptide analysis indicates that a site phosphorylated by PKA resides in a 12-kDa fragment comprising the C terminus of the polypeptide. Studies of phosphorylation performed on homogenates of Xenopus oocytes show that not only endogenous oocyte Na,K-ATPase but also exogenous Xenopus Na,K-ATPase expressed in the oocyte by microinjection of cRNA can be phosphorylated in response to stimulation of oocyte PKA and PKC. In conclusion, these data are consistent with the possibility that the alpha-subunit of Na,K-ATPase can serve as a substrate for PKA and PKC in vivo.     

Purification, immunological and biochemical characterization of Ap4A binding protein from Xenopus laevis oocytes.

Diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) binding protein specifically binds Ap4A. The protein has been purified from Xenopus laevis oocytes and presents an estimated molecular weight of 100,000 by gel filtration. In the first stages of the purification, the Ap4A binding activity is found associated to DNA polymerase alpha-DNA primase, forming heterogeneous high molecular weight complexes. A monoclonal antibody has been prepared against the purified Ap4A binding protein. The antibody partially neutralizes the Ap4A binding activity. Using the immunoblot technique, it has been shown that the antibody is able to recognize either native or SDS-denatured Ap4A binding protein. The monoclonal antibody immunoreacted with a polypeptide of 90,000 which coincides with the molecular weight obtained by gel chromatography and indicates that the native Ap4A binding protein from Xenopus oocytes is probably a monomeric protein.