The Drosophila melanogaster flightless I gene is required for normal cellularization of the syncytial blastoderm. Highly conserved homologues of flightless I are present in Caenorhabditis elegans, mouse, and human. We have disrupted the mouse homologue Fliih by homologous recombination in embryonic stem cells. Heterozygous Fliih mutant mice develop normally, although the level of Fliih protein is reduced. Cultured homozygous Fliih mutant blastocysts hatch, attach, and form an outgrowing trophoblast cell layer, but egg cylinder formation fails and the embryos degenerate. Similarly, Fliih mutant embryos initiate implantation in vivo but then rapidly degenerate. We have constructed a transgenic mouse carrying the complete human FLII gene and shown that the FLII transgene is capable of rescuing the embryonic lethality of the homozygous targeted Fliih mutation. These results confirm the specific inactivation of the Fliih gene and establish that the human FLII gene and its gene product are functional in the mouse. The Fliih mouse mutant phenotype is much more severe than in the case of the related gelsolin family members gelsolin, villin, and CapG, where the homozygous mutant mice are viable and fertile but display alterations in cytoskeletal actin regulation.We are studying the mammalian homologues of a number of Drosophila melanogaster genes concerned with development or behavior, as part of a program aimed at identifying novel mammalian developmental and neurobiological genes. The D. melanogaster flightless I (fliI) gene (4,15,23,24,33) is required for cellularization of the syncytial blastoderm. With severe mutations in fliI, when the contribution of maternal product is eliminated, cellularization is only partial and gastrulation fails (35,44).
SummaryThe product of the flightless I gene is predicted to provide a link between molecules of an as yet unidentified signal transduction pathway and the actin cytoskeleton. Previous work has shown that weak and severe mutations of the flightless I locus in Drosophila melanogaster cause disruption in the indirect flight muscles and in embryonic cellularization events, respectively, indicative of a regulatory role for the flightless I protein in cytoskeletal rearrangements. A C-terminal domain within flightless I with significant homology to the gelsolinlike family of actin-binding proteins has been identified, but evidence of a direct interaction between endogenous flightless I and actin remains to be shown. In the present study, chick, mouse and Drosophila melanogaster embryos have been examined and the localization of flightless I investigated in relation to the actin cytoskeleton. It is shown that flightless I localization is coincident with actin-rich regions in parasympathetic neurons harvested from chicks, in mouse blastocysts and in structures associated with cellularization in Drosophila melanogaster.
In many cellular systems, activation with more than one ligand can produce a cellular response that is greater than the sum of the individual responses to the ligands. This synergy is sometimes referred to as coactivation. In Swiss 3T3 fibroblasts, activation of the epidermal growth factor (EGF) receptor produces a weak induction of DNA synthesis. Insulin has no stimulatory effect on this response. However, in combination, EGF and insulin synergize to cause a large induction of S phase. The underlying cellular biochemistry of this effect has been examined. The data indicate that phospholipase C activation is a major component of agonist‐induced DNA synthesis. In contrast, activation of p70 S6 kinase by single agonists was inversely related to their ability to stimulate DNA synthesis. Therefore, it was examined whether stimulation of Swiss 3T3 cells with insulin causes changes in the subcellular distribution of EGF receptors and phospholipase Cγ1 that could potentially explain the observed synergy or costimulation. It was found that insulin effectively induced the accumulation of EGF receptors on the actin arc of cells without activation of the EGF receptor. In contrast, EGF, when added for several hours, did not cause accumulation of the EGF receptor at this site. However, both EGF and insulin stimulated the accumulation of phospholipase Cγ1 at the actin arc, which was coincident with the EGF receptor in the case of insulin‐ stimulated cells. Therefore, it is suggested that the insulin‐induced coclustering of the EGF receptor with phospholipase Cγ1 at the actin arc may allow for greater efficiency of signal transduction, resulting in the synergy observed for these two hormones in stimulation of DNA synthesis.
The G protein–coupled thrombin receptor can induce cellular responses in some systems by transactivating the epidermal growth factor (EGF) receptor. This is in part due to the stimulation of ectoproteases that generate EGF receptor ligands. We show here that this cannot account for the stimulation of proliferation or migration by thrombin of Swiss 3T3 cells. Thrombin has no direct effect on the activation state of the EGF receptor or of its downstream effectors. However, thrombin induces the subcellular clustering of the EGF receptor at filamentous actin–containing structures at the leading edge and actin arcs of migrating cells in association with other signaling molecules, including Shc and phospholipase Cγ1. In these thrombin-primed cells, the subsequent migratory response to EGF is potentiated. Thrombin did not potentiate the EGF-stimulated EGF receptor phosphorylation. Thus, in Swiss 3T3 cells the G protein–coupled thrombin receptor can potentiate the EGF tyrosine kinase receptor response when activated by EGF, and this appears to be due to the subcellular concentration of the receptor with downstream effectors and not to the overall ability of EGF to induce receptor transphosphorylation. Thus, the EGF receptor subcellular localization which is altered by thrombin appears to be an important determinant of the efficacy of downstream EGF receptor signaling in cell migration.
1. At any one instant, most receptors are now recognized to be able to stimulate multiple signal transduction pathways in a cell when activated by an appropriate hormone. These different signalling pathways appear to allow for distinct cellular responses, such as cell proliferation, differentiation, and shape change. 2. In addition, many different types of cell will possess the same type of receptor. Therefore, for a hormone to be able to transmit differential signals to the various cell types able to respond to it, cells must discriminate the stimulus at some point. Such discrimination would seem to be an absolute requirement to allow a tissue-specific response to an identical initial stimulus. In theory, this specificity could occur at many points in the receptor signal transduction cascade, including cytosolic receptor coupling systems and tissue/cell-specific responsive genes. 3. The present paper summarizes our work and that of others which has determined some of the coupling systems of G-protein-coupled receptors and tyrosine kinase receptors and how these systems may be interacting. 4. In addition to these theoretical considerations, a potential therapeutic strategy underlies the ability of receptors to couple to more than one signal transduction system. If a response to a hormone were, for example, either cell proliferation or cell morphological change or differentiation and separate receptor-coupled signalling systems were responsible for these effects, pharmacological intervention may allow the transfer from one signalling system to another. If such a change allowed a permanent change to the differentiated phenotype, this could potentially form the basis of a signal-based cancer therapy.
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