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Wolbachia is a common maternally inherited bacterial symbiont able to induce crossing sterilities known as cytoplasmic incompatibility (CI) in insects. Wolbachia-modified sperm are unable to complete fertilization of uninfected ova, but a rescue function allows infected eggs to develop normally. By providing a reproductive advantage to infected females, Wolbachia can rapidly invade uninfected populations, and this could provide a mechanism for driving transgenes through pest populations. CI can also occur between Wolbachia-infected populations and is usually associated with the presence of different Wolbachia strains. In the Culex pipiens mosquito group (including the filariasis vector C. quinquefasciatus) a very unusual degree of complexity of Wolbachia-induced crossing-types has been reported, with partial or complete CI that can be unidirectional or bidirectional, yet no Wolbachia strain variation was found. Here we show variation between incompatible Culex strains in two Wolbachia ankyrin repeat-encoding genes associated with a prophage region, one of which is sex-specifically expressed in some strains, and also a direct effect of the host nuclear genome on CI rescue.

Current models for protrusive motility in animal cells focus on cytoskeleton-based mechanisms, where localized protrusion is driven by local regulation of actin biochemistry. In plants and fungi, protrusion is driven primarily by hydrostatic pressure. For hydrostatic pressure to drive localized protrusion in animal cells, it would have to be locally regulated, but current models treating cytoplasm as an incompressible viscoelastic continuum or viscous liquid require that hydrostatic pressure equilibrates essentially instantaneously over the whole cell. Here, we use cell blebs as reporters of local pressure in the cytoplasm. When we locally perfuse blebbing cells with cortex-relaxing drugs to dissipate pressure on one side, blebbing continues on the untreated side, implying non-equilibration of pressure on scales of approximately 10 microm and 10 s. We can account for localization of pressure by considering the cytoplasm as a contractile, elastic network infiltrated by cytosol. Motion of the fluid relative to the network generates spatially heterogeneous transients in the pressure field, and can be described in the framework of poroelasticity.

Nuclear reprogramming is essential during gametogenesis for the production of totipotent zygotes. Here we show that premeiotic female germ cells derived from mouse fetuses as early as 12.5 days post coitum are able to complete meiosis and genomic imprinting in vitro and that these matured oocytes are highly competent in supporting development to full term after nuclear transfer and in vitro fertilization. To our knowledge, this is the first time that complete oogenesis has been successfully accomplished in vitro.

RBM and DAZ/SPGY are two families of genes located on the Y chromosome that encode proteins containing RNA-binding motifs, and both have been described as candidate human spermatogenesis genes. Transmission of deletions from father to son has been observed in the case of DAZ, but neither gene family has been shown to be essential for spermatogenesis in human males. The DAZ/SPGY genes are particularly amenable to a knockout approach, as they are found on the Y chromosome in Old World primates and apes, but in other mammals, they are represented only by an autosomal gene, DAZLA, which is also present in Old World primates and apes. It has also been shown that a Dazla homologue is essential for spermatogenesis in Drosophila. Here we show that Dazla protein is cytoplasmic in male and female germ cells, unlike the nuclear RBM protein. Disruption of the Dazla gene leads to loss of germ cells and complete absence of gamete production, demonstrating that Dazla is essential for the differentiation of germ cells.

Fish melanophore cells aggregate pigment granules at the centre or redisperse them throughout the cytoplasm. The granules move along radial microtubules by means of molecular motors. Cytoplasmic fragments of melanophores organize a radial array of microtubules and aggregate pigment at its centre. Here we report self-centring in microsurgically produced cytoplasmic fragments of black tetra melanophores. We observed rapid (10 min) formation of a radial microtubule array after stimulation of aggregation. Arrangement of microtubules in the fragments returned to random during pigment redispersion. Apparently, formation of the radial array does not depend on a pre-existing microtubule-organizing centre. The array did not form in granule-free fragments nor in fragments treated with inhibitors of the intracellular motor protein cytoplasmic dynein. We conclude that formation of the radial microtubule array is induced by directional motion of pigment granules along microtubules and present evidence that its position is defined by interaction of microtubules with the surface.

Calcium entry into neuronal cells through voltage or ligand-gated ion channels triggers neuronal activity-dependent gene expression critical for adaptive changes in the nervous system. Cytoplasmic calcium transients are often accompanied by an increase in the concentration of nuclear calcium, but the functional significance of such spatially distinct calcium signals is unknown. Here we show that gene expression is differentially controlled by nuclear and cytoplasmic calcium signals which enable a single second messenger to generate diverse transcriptional responses. We used nuclear microinjection of a non-diffusible calcium chelator to block increases in nuclear, but not cytoplasmic, calcium concentrations following activation of L-type voltage-gated calcium channels. We showed that increases in nuclear calcium concentration control calcium-activated gene expression mediated by the cyclic-AMP-response element (CRE), and demonstrated that the CRE-binding protein CREB can function as a nuclear calcium-responsive transcription factor. A second signalling pathway, activating transcription through the serum-response element (SRE), is triggered by a rise in cytoplasmic calcium and does not require an increase in nuclear calcium.

Inwardly rectifying potassium channels conduct ions more readily in the inward than the outward direction, an essential property for normal electrical activity. Although voltage-dependent block by internal magnesium ions may underlie inward rectification in some channels, an intrinsic voltage-dependent closure of the channel plays a contributory, or even exclusive, role in others. Here we report that, rather than being intrinsic to the channel protein, so-called intrinsic rectification of strong inward rectifiers requires soluble factors that are not Mg2+ and can be released from Xenopus oocytes and other cells. Biochemical and biophysical characterization identifies these factors as polyamines (spermine, spermidine, putrescine and cadaverine). The results suggest that intrinsic rectification results from voltage-dependent block of the channel pore by polyamines, not from a voltage sensor intrinsic to the channel protein.

Potassium channels allow potassium ions to flow across the membrane and play a key role in maintaining membrane potential. Recent research has begun to reveal how these channels transport potassium in preference to other ions, how their activity is controlled, and how they are related to other channels.

The intracellular pH of the sea urchin embryo increases 0.3 pH units between 1 and 4 min after fertilisation. The increase in pH is required for initiating development. The increase results from an exchange of extracellular Na+ for intracellular H+.

An increase in cytoplasmic free calcium, [Ca2+]i, is thought to be the trigger for secretory exocytosis in many cells. In blood platelets, large rises in [Ca2+]i can cause secretion and calcium has been regarded as the final common activator not only for secretion but also for shape-change and aggregation. We have shown that while thrombin and platelet-activating factor (PAF) normally elevate [Ca2+]i, they can also stimulate shape-change and secretion even when the [Ca2+]i rise is suppressed. The present results strongly implicate diacylglycerol, produced by stimulus-dependent breakdown of phosphoinositide, in this calcium-independent activation. Exogenous diacylglycerol activates a protein kinase (C-kinase) in platelets as do PAF, thrombin and collagen. 12-O-tetradecanoyl phorbol-13-acetate (TPA) also activates C-kinase and is a potent stimulus for secretion and aggregation. It is shown here that the exogenous diacylglycerol 1-oleoyl-2-acetyl-glycerol (OAG) and TPA evoke similar secretion and aggregation without elevating [Ca2+]i above the basal level of 0.1 microM. The pattern of secretion resembles that produced by collagen and thrombin when [Ca2+]i remains at basal levels. Modest increases in [Ca2+]i, insufficient to stimulate secretion, markedly accelerate the responses to TPA and OAG.

The segmental pattern of insect embryos depends on influences from morphogenetic centres near each of the egg poles. In Drosophila, maternal effect mutations are known that impair the normal function of each centre. Injection of wild-type cytoplasm into mutant eggs has revealed that morphogenetic signals localized at the anterior and posterior pole of eggs can be transplanted. We show here that these activities can also be detected during oogenesis. Posterior activity can be recovered at an early stage (stage 10, ref. 5) from the oocyte-nurse cell complex, but anterior activity can only be detected in the mature oocytes (stage 14). We conclude that the bicoid-dependent anterior signal, although produced by the nurse cells, does not become active before it is localized to the anterior egg pole, whereas posterior activity can be detected in the nurse cells before, and therefore independently of, its localization to the posterior egg pole.

Deletion of specific nucleotides at either end of the long terminal repeat of the avian retrovirus, spleen necrosis virus, results in replication-competent but integration-defective virus. This result supports two conclusions: (1) the 5-base pair terminal inverted repeats and three to seven adjacent nucleotides are required for integration; (2) integration of retrovirus DNA is not required for retrovirus gene expression.

It is known that glucose-induced depolarization of pancreatic B-cells is due to reduced membrane K+-permeability and is coupled to an increase in the rate of glycolysis, but there has been no direct evidence linking specific metabolic processes or products to the closing of membrane K+ channels. During patch-clamp studies of proton inhibition of Ca2+-activated K+ channels [GK(Ca)] in B-cells, we identified a second K+-selective channel which is rapidly and reversibly inhibited by ATP applied to the cytoplasmic surface of the membrane. This channel is spontaneously active in excised patches and frequently coexists with GK(Ca) channels yet is insensitive to membrane potential and to intracellular free Ca2+ and pH. Blocking of the channel is ATP-specific and appears not to require metabolism of the ATP. This ATP-sensitive K+ channel [GK(ATP)] may be a link between metabolism and membrane K+-permeability in pancreatic B-cells.

The mechanisms by which growth factors stimulate metabolism and cell proliferation are largely unknown. Recent evidence suggests that mitogens rapidly activate a Na+/H+ exchange mechanism in the plasma membrane of their target cells, implicating cytoplasmic pH (pH1) as a potential 'messenger'. Indeed, growth stimulation of quiescent fibroblasts leads to intracellular alkalinization at approximately 1 h after mitogen addition, as measured by weak-acid distribution methods. We have used an internalized fluorescent pH1 indicator to examine the pH1-regulating mechanisms in diploid human fibroblasts and to obtain the first continuous pH1 recordings of the response to growth factors. We report here that (1) pH1 in human fibroblasts is controlled by a membrane-bound Na+/H+ exchanger, which rapidly restores pH1 after an acute cytoplasmic acidification, and (2) epidermal growth factor (EGF) and serum factors induce a rapid and persistent elevation of pH1 by modifying the pH1 sensitivity of the Na+H+ exchanger. We conclude that in addition to having a basic role in pH1 regulation, Na+/H+ exchange may function as a transmembrane signal transducer in the action of growth factors.

Somatic cell hybridization is a valuable tool for investigation the control of gene expression in eukaryotic cells. Studies of hybrid cells, heterokaryons, reconstructed cells and cybrids (cytoplasmic hybrids) have suggested that cytoplasmic factors may be involved in this regulatory process. Unfortunately, studies of this kind usually require that hybrid or modified cells be maintained for some time in a selective environment during which chromosomal losses or other changes may modify the genetic functions of the cells and thus vitiate conclusions about the mechanism of gene regulation. We report here the preparation of cybrids between enucleated mouse fibroblasts (Cl-1-D) and differentiated rat hepatoma cells (Fao) and the use of a combination of histological techniques to identify these modified cells early after fusion without the use of selective media. We found that albumin production in most cybrids was suppressed (extinguished) at 12-20 h after fusion but was restored by 48 h. These results suggest that there is a cytoplasmic factor in the fibroblast which exerts negative control over expression of the albumin gene, but which in the absence of the fibroblast nucleus, is not renewed and therefore short-lived.

In several types of locomoting cells, active rearward transport of particles on the cell surface has been observed and correlated with motility. No forward transport of particles has previously been reported, however. Here we report rapid forward transport of concanavalin A-coated gold particles on the dorsal surfaces of lamellipodia of fish epidermal keratocytes. These movements are active, not diffusive, and more rapid than either rearward particle transport or the rate of cell locomotion. We observed forward transport in migrating, but not in stationary cells, and could block the movement by treatment with cytochalasin D. These studies demonstrate for the first time that small numbers of glycoproteins can be actively transported on the surface of the cell to the front of the lamellipodium. We suggest that this mechanism transports proteins involved in cell locomotion, such as proteins necessary for adhesion, and could also produce an extensile force.

A protein synthesised during oogenesis seems to be essential for the activation, during blastulation, of the nuclear genes essential for gastrulation and organogenesis. Nuclear transplantation experiments show that this interaction between the protein and the blastula nucleus produces a heritable state of nuclear activation.

When two strains of Culex pipiens (s.l.) of different geographical origin are cross-mated, the cross is frequently sterile in one or both directions. Such incompatibility is said to be cytoplasmic because the crossability of a strain is determined by its maternal lineage. The incompatibility is caused in some way by infection with a rickettsia-like bacterial symbiote, as removal of the symbiote abolishes the incompatibility. In compatibility has not been observed in crosses of American strains of C. pipiens. On the other hand, most workers in other parts of the world who have crossed C. pipiens strains have noticed incompatibility, although there are no reports of incompatible egg rafts being collected in the field. We now report incompatibility in crosses of sympatric American strains of C. pipiens and the collection of incompatible egg rafts in the field.

Parthenogenetic mouse embryos pose an interesting problem in the study of early mammalian development. Haploid or diploid parthenogenones, resulting from spontaneous or experimental activation of unfertilized eggs, will undergo apparently normal preimplantation development but die in the early post-implantation stages. However, in aggregation chimaeras with fertilized embryos, parthenogenetic embryos have the ability to differentiate into many tissue types, including gametes which can give rise to normal offspring. Furthermore, it has been reported that viable young were obtained from the transfer of inner cell-mass nuclei of parthenogenetic blastocysts to enucleated fertilized eggs. These observations suggest that sperm have some additional role, apart from restoring a complete genome, that is necessary for normal development. To investigate whether sperm-related modifications to the egg cytoplasm are important, we have used an efficient nuclear transfer technique in which a complete karyoplast, comprised of pronuclei, surrounding cytoplasm and a portion of the egg plasma membrane, is transferred utilizing Sendai virus membrane fusion. Embryos produced by the transfer of pronuclei from diploid parthenogenetic eggs to enucleated fertilized eggs died very soon after implantation, whereas viable young were obtained from the transfer of fertilized egg pronuclei into enucleated parthenogenetic eggs. This shows that the death of parthenogenones is not due to a lack of cytoplasmic factors from the sperm.

The maternally transmitted antigen (Mta) is expressed on the cells of most strains of mice. It is a medial histocompatibility antigen, that is, it is recognized by unrestricted cytotoxic T lymphocytes as are major H antigens, but unlike these it is a weak transplantation antigen and does not itself restrict the T-cell recognition of minor H antigens. All other medial H antigens are encoded by genes closely linked to the major histocompatibility complex, H-2 in the mouse. By contrast, Mta appeared to follow extrachromosomal, maternal inheritance. Several substrains of NZB, NZO and non-inbred European NMRI mice are Mta-negative. Females of these strains bear only Mta- offspring, while females of the inbred Mta+ strains bear only Mta+ offspring. Repeated backcrossing from Mta+ females to NZB or NMRI males has shown that, given the right cytoplasmic genes, the chromosomal genes of these Mta- strains permit expression of Mta2. As the Mta type of a mouse cannot be influenced by embryo transfer or foster nursing, we concluded that it was determined by a cytoplasmic factor (Mtf), transmitted through the egg. We now show that a gene, Hmt, closely linked to the H-2 complex, is also required for expression of Mta.

Cellular proliferative activity has previously been determined by measuring the incorporation of radiolabelled nucleotides or by visual inspection of cellular morphology. Although two flow cytometric methods have recently been developed which can distinguish cycling from non-cycling cells, both have serious disadvantages. One method requires uptake of a substantial amount of BUdR, limiting its usefulness for in vitro systems. The other method utilizes RNA/DNA content differences but its successful application has proved cell-type dependent. We have now used the findings that the cell membrane is more highly polarized in resting than in proliferating cells and that cyanine dyes carrying a delocalized positive charge enter live cells to an extent that depends on the cell membrane potential, to develop a method of distinguishing between cycling and non-cycling cells. The greater the membrane polarization, the greater is the concentration of dye within the cell. At high concentrations, the dye molecules aggregate and their fluorescence is quenched. Thus, for a given external dye concentration, cells of different membrane potential would accumulate different amounts of fluorescent (non-aggregated) dye. Using fibroblasts in culture conditions chosen to provide various models of cycling and non-cycling cells, we found that fluorescence intensity with the dye, 3,3'-diheptyloxycarbocyanine (Di-O-C,(3)) was consistently greater in the former than the latter.

Measurements with the Ca2+ -sensitive photoprotein aequorin show that locomotion in the amoeba Chaos carolinense occurs without changes in the aequorin signal and that not more than 0.025% of the cytoplasm can exist at the micromolar threshold concentration for contraction. The results do not support the hypothesis that cytoplasmic streaming is under the control of changes in the cytoplasmic free Ca2+ concentration.

The vertebrate visual system can operate over a large range of light intensities. This is possible in part because the sensitivity of photoreceptors decreases approximately in inverse proportion to the background light intensity. This process, called photoreceptor light adaptation, is known to be mediated by a diffusible intracellular messenger, but the identity of the messenger is still unclear. There has been considerable speculation that decreased cytoplasmic Ca2+ concentration (Cai2+) may play a role in light adaptation, and recent experiments in which Ca2+ buffer was incorporated into rod-cells have supported this notion. The extent of the contribution of calcium, however, remains unresolved. We now show that light-dependent changes in sensitivity in amphibian photoreceptors can be abolished by preventing movements of Ca2+ across the outer-segment plasma membrane. These experiments demonstrate that light adaptation in photoreceptors is mediated in cones primarily, and in rods perhaps exclusively, by changes in Cai2+.

Mature oocytes (unfertilized eggs) are arrested at definite cell-cycle stages which vary from species to species. In frogs and mammals, the oocytes are arrested at the second metaphase of meiosis whereas in echinoderms they are blocked later, at the pronucleus stage. What causes the maturing oocytes to stop at some point in the cell cycle is not entirely clear. In frogs, the metaphase arrest seems to be maintained by a cytostatic factor. In echinoderms, which stop at interphase, no such a factor has so far been found. The fertilization process, beyond the introduction of paternal chromosomes, releases the oocyte from cell-cycle arrest and provides a functional centrosome to replace the endogenous centrosome which is apparently lost during oogenesis in most species. Several lines of evidence suggest that release from cell-cycle arrest is mediated by a Ca2+ burst which is associated with fertilization, and it is known that the functional centrosome provided by the sperm is necessary for mitotic spindle formation and cleavages. We report here that microinjection of purified human centrosomes into mature starfish oocytes is sufficient to release them from arrest at interphase and to support many cleavages leading to the occasional formation of normal embryos. In this species centrosome induced re-entry into the cell cycle does not require a transient calcium burst nor does it require intact microtubules.

A mouse major histocompatibility antigen (H-2) gene, encoding a novel H-2Ld molecule lacking its intracytoplasmic domain, has been constructed and introduced into mouse L-cells. The novel H-2 molecule is found on the surface of the transfected cells at the same level as L-cells transfected with the native H-2Ld gene. Allo- and influenza-specific cytotoxic T lymphocytes can recognize the truncated H-2 gene product nearly as efficiently as the normal H-2Ld gene product. However, vesicular stomatitis virus-specific cytotoxic T lymphocytes recognize the truncated H-2Ld molecule less efficiently than the complete H-2Ld product. The rate of capping of the truncated H-2Ld molecule was investigated and found to be the same as that of the complete H-2Ld gene product.

Insulin regulates the synthesis of several proteins in a variety of tissues. Before techniques were available to quantify the amount of specific mRNAs, insulin was thought to regulate the synthesis of proteins by influencing the rate of translation of a fixed amount of mRNA. A very different interpretation is called for by experiments which show that insulin alters the amount of several specific mRNAs, but little is known about the mechanism. Insulin decreases the rate of synthesis of the critical gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) in both liver and H4IIE heptoma cells. We recently showed that insulin acts directly on H4IIE cells to decrease mRNAPEPCK activity without any other hormone intermediaries. This effect is mediated by the insulin receptor and occurs at insulin concentrations which are well within the physiological range range (10(-12)--10(-9) M). Here we extend these studies to show that insulin specifically inhibits transcription of the PEPCK gene. This inhibition results in a rapid decrease in the concentration of nuclear PEPCK transcripts which is followed, in turn, by a proportionate decline in cytoplasmic mRNAPEPCK and synthesis of the protein.

We report here electrophysiological and ion distribution results obtained with mechanically skinned skeletal muscle fibres which indicate that a Donnan equilibrium, characterized by large ionic concentration and electrical potential differences, is established between the myofibrillar space and the rest of the sarcoplasm surrounding the myofibrils. The more negative electrical potential within the myofibrils (-15 to -29 mV for conditions similar to those in vivo) can strongly influence: (1) intracellular Ca2+ movements associated with contraction; (2) the location of sarcoplasmic enzymes, their substrates and products of reaction; (3) measurements of the electrical potential difference between the extra- and intracellular space and estimation of relative membrane permeabilities for certain ions.