Fertilization and pronuclear migration in the ctenophore, Beroe ovata. Video supplement.
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Animals↗
Biomedical subjects
C Sardet
Publications and source records attributed to C Sardet.
[Role of the multifunctional Trio protein in the control of the Rac1 and RhoA gtpase signaling pathways].
The small GTPases Cdc42, Rac and RhoA have important regulatory roles in mediating cytoskeletal rearrangements, MAP kinase cascades and induction of G1 cell cycle progression. The activity of the GTPases is regulated by guanine nucleotide exchange factors (GEFs) which accelerate their GDP/GTP exchange rate, and thereby activate them. All the GEFs for the Rho-GTPases family share two conserved domains: the DH domain (for Dbl-homology domain) responsible for the enzymatic activity, and the PH domain, probably responsible for the proper localization of the molecule. Trio is a multifunctional protein that is comprised of two functional Rho-GEFs domains and a serine/threonine kinase domain. We have shown in vitro and in vivo that the first GEF domain (GEFD1) activates Rac1, while the second GEF domain (GEFD2) acts on RhoA. Moreover, the co-expression of both domains induces simultaneously the activation of both GTPases. To our knowledge, this is the first example of a member of the Rho-GEF family, that contains two functional exchange factor domains, with restricted and different specificity. We are currently investigating how these GEF domains are activated, by addressing the role of the PH domains in GTPases activation by Trio. We have shown that: 1) the PH1 of Trio is necessary for Rac activation by the GEFD1; 2) the PH1 of Trio targets the molecule to the cytoskeleton; 3) the GEFD1 domain of Trio binds, in a two-hybrid screen, the actin binding protein filamin. These data suggest that the PH1 targets Trio to the cytoskeleton close to Rac and its effectors, probably via interaction with the actin-binding protein filamin, consistent with a role of Trio in actin cytoskeleton remodeling.
3T3 Cells↗
Formation and localization of cytoplasmic domains in leech and ascidian zygotes.
Leech and ascidian embryos are well suited for the study of certain developmental processes. Although leeches and ascidians belong to different bilateralia groups (protostomes and deuterostomes, respectively) they share important developmental features and, in particular, the determinate character of their embryogenesis. In both types of embryos this property is related to the presence of specific cytoplasmic domains that are selectively allocated to different blastomeres during cleavage. In this review leech and ascidian eggs and zygotes are compared in terms of the structure of these cytoplasmic domains and of the cellular mechanisms involved in their formation and localization. During meiosis the zygote of leeches and ascidians undergo stereotypic actin-dependent contraction movements related to both the emission of the polar bodies and the formation and relocalization of cytoplasmic domains. After completion of meiosis, during first interphase, monaster microtubules nucleated from the sperm-derived centrosome play a key role in pronuclear migration. In addition, these astral microtubules direct the relocalization of cytoplasmic domains and the translocation and accumulation of organelles in the interior of the zygote. Microtubules and microfilaments, on the other hand, are involved in cortical reorganizations and organelle translocations in both zygote species during interphase and cleavage divisions. In the case of leech zygotes, this process leads to formation of characteristic polar cytoplasmic domains called teloplasms. These domains are selectively inherited by teloblasts, precursor stem cells of ectodermal and mesodermal tissues in the leech embryo. In the ascidian zygote, the cytoplasmic movements observed during interphase and mitosis lead to relocalization of the bulk of a mitochondria-rich domain, called the myoplasm, along with an endoplasmic reticulum-rich domain towards the future posterior pole of the embryo. The myoplasm is inherited by a subset of posterior blastomeres committed to become the primary muscle cells of the ascidian tadpole.
Animals↗
The two intracellular Ca2+ release channels, ryanodine receptor and inositol 1,4,5-trisphosphate receptor, play different roles during fertilization in ascidians.
Fertilization in the ascidians triggers an activation wave of calcium release followed by intracellular calcium oscillations synchronous with periodic membrane potential excursions during the completion of the meiotic cell cycle. Fertilization also causes a fast decrease in the egg plasma membrane depolarization-activated calcium current and a large increase in capacitance thought to represent membrane addition to the egg surface. We have analyzed the temporal and causal relationships between these changes in the eggs of Phallusia mammillata using whole-cell patch-clamp recording while simultaneously imaging calcium with fura-2 dextran. We have defined the role of ryanodine receptor (RyR) and InsP3 receptor (InsP3R) during fertilization and meiosis by looking at the effects of InsP3, cyclic ADP ribose (cADPR), and ryanodine in perfused oocytes. We show that InsP3 (10 microM perfused through the patch pipette) is able to trigger sustained oscillations in intracellular calcium concentration in unfertilized oocytes, resembling those recorded in fertilized egg completing meiosis. In addition the sustained oscillations resulting from InsP3 perfusion in unfertilized oocytes are sufficient to cause the emission of both polar bodies. In contrast, ryanodine or cADPR never trigger detectable calcium signal in perfused oocytes. Instead, nanomolar concentrations of ryanodine or cADPR cause a capacitance change, implying a net insertion of membrane to the oocyte surface, and trigger a fast decrease in the depolarization-activated calcium current. Both changes are similar to the changes in conductance and capacitance naturally observed following fertilization. These effects, although not associated with measurable calcium signals, are abolished by coperfusion of the calcium chelator BAPTA. In contrast to ryanodine or cADPR, sustained perfusion of the oocyte with nanomolar concentrations of InsP3 causes no capacitance change and a slow and moderate decrease in calcium current. Our observations on inseminated patch-clamped eggs further indicate that membrane insertion, which starts 15-20 sec after the onset of the membrane conductance change at fertilization, can be altered by interfering with the RyR. Our results imply that, in ascidians, as in some mammals, RyR and InsP3R play distinct roles during fertilization.
Animals↗
Interaction between Cdc37 and Cdk4 in human cells.
Using the yeast two-hybrid system we have identified novel potential Cdk4 interacting proteins. Here we described the interaction of Cdk4 with a human homologue of the yeast Drosophila CDC37 gene products. Cdc37 protein specifically interacts with Cdk4 and Cdk6, but not with Cdc2, Cdk2, Cdk3, Cdk5 and any of a number of cyclins tested. Cdc37 is not an inhibitor nor an activator of the Cdk4/cyclin D1 kinase, while it appears to facilitate complex assembly between Cdk4, and cyclin D1 in vitro. Cdc37 competes with p16 for binding to Cdk4, suggesting that p16 might exert part of its inhibitory function by affecting the formation of Cdk4/cyclin D1 complexes via Cdc37.
Amino Acid Sequence↗
Regulation of cyclin E transcription by E2Fs and retinoblastoma protein.
Cyclin E is critical for the advance of cells through the G1 phase of their growth cycle. Transcription of the cyclin E gene is known to be cell cycle-dependent. We have shown previously that mRNA levels of cyclin E are regulated positively by mitogens and negatively by TGF-beta. Much circumstantial evidence implicates both E2F transcription factors and the retinoblastoma protein (pRB) in the control of cyclin E expression. However, the molecular basis of this control has remained unclear. We report here the cloning of the cyclin E promoter and the identification of several putative E2F binding sites within the promoter sequence. We have found that cell cycle regulation of cyclin E transcription is mediated by E2F binding sites present in the promoter. The activity of this promoter can be regulated negatively by pRB. Our results suggest the operation of a positive-feedback loop in late G1 that functions to ensure continued cyclin E expression and pRB inactivation.
3T3 Cells↗
A human protein with homology to Saccharomyces cerevisiae SNF5 interacts with the potential helicase hbrm.
In yeast, the SNF/SWI complex is involved in transcriptional activation of several inducible promoters, possibly by causing a local modification of the chromatin structure. Recently, two human homologues of the SNF2/SWI2 protein have been isolated, hbrm and BRG-1. In addition, a complex containing one of the SNF2/SWI2 homologues and having an in vitro activity similar to the yeast complex has been partially purified from HeLa cells. Here we describe the characterization of a cDNA encoding a human nuclear protein containing a large domain of homology with SNF5, another member of the yeast SNF/SWI complex. This protein can be co-immunoprecipitated with hbrm and the interaction between the two proteins is dependent on the region conserved between the human and the yeast SNF5. These findings suggest that the cDNA we have cloned encodes one of the members of the human SNF/SWI complex.
Amino Acid Sequence↗
E2F-4 and E2F-5, two members of the E2F family, are expressed in the early phases of the cell cycle.
The E2F transcription factors play a role in regulating the expression of genes required for cell proliferation. Their activity appears to be regulated by association with the retinoblastoma protein (pRb) and the pRb-related proteins p107 and p130. In vivo, pRb is found in complex with a subset of E2F components--namely, E2F-1, E2F-2, and E2F-3. Here we describe the characterization of cDNAs encoding two unusual E2Fs, E2F-4 and E2F-5, each identified by the ability of their gene product to interact with p130 in a yeast two-hybrid system. E2F-4 and -5 share common sequences with E2F-1, E2F-2, and E2F-3 and, like these other E2Fs, the ability to heterodimerize with DP-1, thereby acquiring the ability to bind an E2F DNA recognition sequence with high affinity. However, in contrast to E2F-1, E2F-4 and E2F-5 fail to bind pRb in a two-hybrid assay. Moreover, they show a unique pattern of expression in synchronized human keratinocytes: E2F-4 and E2F-5 mRNA expression is maximal in mid-G1 phase before E2F-1 expression is detectable. These findings suggest that E2F-4 and E2F-5 may contribute to the regulation of early G1 events including the G0/G1 transition.
Amino Acid Sequence↗
Function and characteristics of repetitive calcium waves associated with meiosis.
BACKGROUND: Internal calcium waves and oscillations are now recognized as universal features of cellular activation, but their exact role remains uncertain. In mammalian and ascidian eggs, a large, sperm-triggered calcium activation wave crosses the egg at fertilization, followed by a series of periodic increases in intracellular calcium concentration ([Ca2+]i). We have previously shown that, in eggs of the ascidian Phallusia mammillata, these periodic, post-activation [Ca2+]i increases are in the form of waves, the origin of which relocalizes to a pacemaker region, and that they stop seconds before the completion of meiosis. RESULTS: We show here that the origin of the first one to four post-activation calcium waves in P. mammillata eggs transfers progressively from the site of sperm entry, usually in the animal hemisphere, towards an endoplasmic reticulum (ER)-rich contraction pole in the vegetal hemisphere, a process that takes about five minutes. Once the origin of these repetitive post-activation calcium waves has reached the contraction pole, all subsequent calcium waves originate from the domain of ER concentrated there, which acts as a pacemaker. The first few post-activation calcium waves are faster than the activation wave and, like the activation wave, they propagate homogeneously throughout the cytoplasm. Approximately five to ten minutes after fertilization, the post-activation calcium waves begin to propagate preferentially in the egg cortex. By manipulating intracellular calcium levels with caged inositol 1,4,5 trisphosphate (InsP3) and a competitive inhibitor of InsP3-induced calcium release, we show that the activation wave induced by the sperm is sufficient to induce extrusion of the first polar body, but that additional [Ca2+]i increases are necessary for completion of the second meiotic division. However, periodic calcium waves per se do not seem to be strictly necessary for the completion of meiosis, as a persistent and homogeneous increase in calcium, induced by the calcium ionophore ionomycin, is sufficient to cause second polar body formation and allow completion of meiosis on time. CONCLUSION: These results clearly show that, in the ascidian egg, post-activation calcium waves are required to complete meiosis. They also show that following a period of progressive relocalization of the wave origin, which lasts approximately five minutes, an ER-rich domain at the contraction pole finally becomes a pacemaker from which the calcium waves originate. Once their origin becomes stably localized, the calcium waves begin to propagate preferentially around the cortex of the egg rather than throughout the egg cytoplasm.
Animals↗
Cytoskeleton and ctenophore development.
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Animals↗
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.
Acetylcholine↗
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.
Animals↗
Characteristics of pronuclear migration in Beroe ovata.
In the large eggs (approximately 1 mm) of the ctenophore Beroe ovata, female pronuclei migrate long distances to join stationary male pronuclei in the peripheral cytoplasm that surrounds the yolky interior. We have investigated the mechanism of nuclear migration using time lapse video recording, automated image analysis, visualization of microtubules by immunofluorescence and rhodamine-tubulin injection, and electron microscopy. Female pronuclei migrated at average speeds of 0.2 microns/sec, and were found to show periodic oscillations in velocity. Alternating phases of acceleration and deceleration occurred with an average periodicity of 235 seconds covering distances of 47 microns (about 3 times the nuclear diameter). Migration velocities and velocity oscillations were similar in fertilized and unfertilized eggs; however, changes in migration direction were much more frequent in unfertilized eggs. Characteristic deformations of the pronuclear membrane and occasional rotation of the nuclear contents were observed during migration. Inhibitor studies indicated that microtubules are required for nuclear migration. In fertilized eggs the top of the nucleus was found to move through the dense layer of aligned sperm aster microtubules. The frequent changes in direction of pronuclear migration in unfertilized eggs reflect the random organization of the microtubule layer in the absence of sperm derived centrosomes. Densely packed endoplasmic reticulum was found intermeshed with sperm aster microtubules and connected extensively with the nuclear membrane during migration. Most nuclear pores were grouped in an infolding of the nuclear membrane. We suggest that in fertilized eggs the female pronucleus is transported to the minus ends of sperm aster microtubules using motor molecules attached either to the outer nuclear membrane and/or to the network of connecting ER.
Animals↗
Cytoplasmic domains in eggs.
One way of organizing the body plan of a developing embryo is to establish domains in the egg with distinct compositions in defined spatial relationships to one another: when the egg divides up, these domains segregate preferentially to certain regions of the embryo and influence their development. In this review we discuss the nature, formation and reorganization of distinguishable domains in various eggs.
Journal Article↗
Immunolocalization of anion exchanger AE2 and cation exchanger NHE-1 in distinct adjacent cells of gastric mucosa.
The gastric mucosa secretes both protons and bicarbonate. The molecular identity of the H(+)-K(+)-ATPase, which mediates acid secretion, has long been known, but the other components of the secretory machinery and their cellular disposition are less well characterized. This study identifies and localizes in rat and rabbit gastric mucosa a chloride-bicarbonate exchanger protein and a Na(+)-H+ exchanger protein. The previously described band 3-related protein of the parietal cell has been identified by isoform-specific antibodies as anion exchanger (AE) 2 and localized to the basolateral membranes of the parietal cells. The Na(+)-H+ exchanger protein NHE-1 was located in the basolateral membranes of the mucous neck cells, interdigitated between the parietal cells of the gastric glands and in the basolateral membranes of the surface mucous cells. Neither transporter protein was abundantly expressed deep in the gland, where most of the pepsinogen cells reside. Carbonic anhydrase II (CA II) was expressed at higher abundance in the surface mucous cells and mucous neck cells, which expressed NHE-1, than in the parietal cells, which expressed AE2. The morphological evidence identified AE2 as a major parietal cell anion exchanger, whereas NHE-1 and CA II colocalized in mucous neck, chief, and surface mucous cells. We propose that all three of these cell types contribute to gastric bicarbonate secretion.
Animals↗
Role of cytoplasmic domain of the Na+/H+ exchanger in hormonal activation.
The growth factor-activated Na+/H+ exchanger is regulated by numerous stimuli, including polypeptide hormones, phorbol esters, cell acidity, and cell shrinkage. To determine whether this regulation occurs at a common site on the cytoplasmic domain of the Na+/H+ exchanger, we microinjected polyclonal antibodies (RP1-c28) to the C-terminal 157 amino acids of the molecule and measured cell pH changes after application of a variety of stimuli known to activate the Na+/H+ exchanger. Microinjection of approximately 10 fg of RP1-c28 antibody, but not control IgG, into single cultured fibroblasts blocked subsequent activation of the exchanger by both endothelin and alpha-thrombin. In contrast, microinjected RP1-c28 did not prevent activation of Na+/H+ exchange by phorbol esters, consistent with the observation that both endothelin-1 and alpha-thrombin retained the ability to activate exchange activity in protein kinase C-depleted cells. Finally, activation of Na+/H+ exchange by both cell acidity and osmotic shrinkage was also unaffected by microinjected RP1-c28 antibody. These data indicate that activation of Na+/H+ exchange by endothelin-1 and alpha-thrombin is mechanistically distinct both from activation by protein kinase C and activation by physical factors and probably occurs at a separate site on the exchanger molecule.
Amiloride↗
Net calcium and acid release at fertilization in eggs of sea urchins and ascidians.
Sea urchin eggs lose about 10-30% of their total calcium content upon fertilization. We have investigated the mechanism of this calcium-loss with an ion-selective vibrating probe system. Upon fertilization of Arbacia punctulata and Lytechinus pictus eggs we could measure a calcium efflux signal with an average duration of 204 +/- 26 s and 146 +/- 46 s, respectively. Measurements of hydrogen ion signals in normal and in low sodium media showed that the release of cortical vesicle material from these eggs lasts for about 30 and 50 s, respectively. The data indicate that most of the calcium that is lost from sea urchin eggs originates from the cytosol in which it is released during fertilization and then pumped out through the plasma membrane. Calcium loss due to cortical granule release accounts for less than 14% of the total loss measured. We also measured a substantial post-fertilization calcium efflux in eggs of Phallusia mammilata, with an average duration of 265 +/- 18 s followed by smaller periodic effluxes that corresponded to oscillations in the [Ca2+]i during contractile waves in these eggs. These data, together with the lack of cortical granules in ascidian eggs, indicate that Phallusia eggs also pump out a substantial amount of calcium through the plasma membrane after fertilization.
Animals↗
Polarity and reorganization of the endoplasmic reticulum during fertilization and ooplasmic segregation in the ascidian egg.
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.
Animals↗