The Experts below are selected from a list of 2616 Experts worldwide ranked by ideXlab platform
Anne J Ridley - One of the best experts on this subject based on the ideXlab platform.
-
rac1 deficient macrophages exhibit defects in cell spreading and Membrane Ruffling but not migration
2004Co-Authors: Claire M Wells, Marita Walmsley, Steen K T Ooi, Victor L J Tybulewicz, Anne J RidleyAbstract:Rac GTPases are activated by extracellular stimuli and contribute to cellular responses including cytoskeletal changes and cell migration. Dominant-negative Rac1 has been used to implicate Rac GTPases in these responses, but which of the three mammalian Rac isoforms it inhibits is not known. We show that mouse bone marrow-derived macrophages express Rac1, low levels of Rac2 but not Rac3. As Rac1-null mice die early in development, we have used mice with a loxP-flanked allele of Rac1 and the type I interferon-inducible Mx1-Cre transgene to address for the first time the specific role of Rac1 in cell motility. Bone marrow-derived macrophages isolated from mice treated with polyIC to induce interferon lack detectable Rac1, and there is no compensatory increase in Rac2 or Cdc42 expression. Rac1-deficient macrophages have an altered morphology: they are significantly more elongated than control cells and have a reduced adhesive area. Re-expression of Rac1 reverts the morphology to that of control cells. Loss of Rac1 reduces but does not completely prevent Membrane Ruffling in response to CSF-1. However, Rac1-deficient macrophages show normal migration and chemotaxis. Thus in macrophages Rac1 is primarily responsible for regulating cell morphology, contributes to Membrane Ruffling, but is not required for migration.
-
role of phosphoinositide 3 oh kinase in cell transformation and control of the actin cytoskeleton by ras
1997Co-Authors: Pablo Rodriguezviciana, Anne J Ridley, Patricia H Warne, Asim Khwaja, Barbara M Marte, Darryl J Pappin, Michael D Waterfield, Julian DownwardAbstract:The pathways by which mammalian Ras proteins induce cortical actin rearrangement and cause cellular transformation are investigated using partial loss of function mutants of Ras and activated and inhibitory forms of various postulated target enzymes for Ras. Efficient transformation by Ras requires activation of other direct effectors in addition to the MAP kinase kinase kinase Raf and is inhibited by inactivation of the PI 3-kinase pathway. Actin rearrangement correlates with the ability of Ras mutants to activate PI 3-kinase. Inhibition of PI 3-kinase activity blocks Ras induction of Membrane Ruffling, while activated PI 3-kinase is sufficient to induce Membrane Ruffling, acting through Rac. The ability of activated Ras to stimulate PI 3-kinase in addition to Raf is therefore important in Ras transformation of mammalian cells and essential in Ras-induced cytoskeletal reorganization.
-
3bp 1 an sh3 domain binding protein has gap activity for rac and inhibits growth factor induced Membrane Ruffling in fibroblasts
1995Co-Authors: Piera Cicchetti, Anne J Ridley, Yi Zheng, R A Cerione, David BaltimoreAbstract:Abstract The SH3 binding protein, 3BP-1, was originally cloned as a partial cDNA from an expression library using the Abl SH3 domain as a probe. In addition to an SH3 binding domain, 3BP-1 displayed homology to a class of GTPase activating proteins (GAPs) active against Rac and Rho proteins. We report here a full length cDNA of 3BP-1 which extends the homology to GAP proteins previously noted. 3BP-1 functions in vitro as a GAP with a specificity for Rac-related G proteins. Microinjection of the 3BP-1 protein into serum-starved fibroblasts produces an inhibition of platelet-derived growth factor (PDGF)-induced Membrane Ruffling mediated by Rac. Co-injection of 3BP-1 with an activated Rac mutant that is unresponsive to GAPs, counter-acts this inhibition. 3BP-1 does not show in vitro activity towards Rho and, in agreement with this finding, microinjection of 3BP-1 into fibroblasts has no effect on lysophosphatidic acid (LPA)-induced stress fiber assembly mediated by Rho. Thus 3BP-1 is a new and specific Rac GAP that can act in cells to counter Rac-mediated Membrane Ruffling. How its SH3 binding site interacts with its GAP activity remains to be understood.
-
Membrane Ruffling and signal transduction
1994Co-Authors: Anne J RidleyAbstract:One of the earliest structural changes observed in cells in response to many extracellular factors is Membrane Ruffling: the formation of motile cell surface protrusions containing a meshwork of newly polymerized actin filaments. It is becoming clear that actin reorganization is an integral part of early signal transduction pathways, and that many signalling molecules interact with the actin cytoskeleton. The small GTP-binding protein Rac is a key regulator of Membrane Ruffling, and proteins that can regulate Rac activity, such as Bcr, are likely to act on this signalling pathway. In addition, several previously characterized signal transducing molecules are implicated in the Membrane-Ruffling response, including Ras, the adaptor protein Grb2, phosphatidyl inositol 3-kinase, phospholipase A2 and phorbol ester-responsive proteins. Changes in polyphosphoinositide metabolism and intracellular Ca2+ levels may also play a role. A number of actin-binding and organizing proteins localize to Membrane ruffles and are potential targets for these signal transducing molecules.
-
the small gtp binding protein rac regulates growth factor induced Membrane Ruffling
1992Co-Authors: Anne J Ridley, Hugh Paterson, Caroline L Johnston, Dagmar Diekmann, Alan HallAbstract:The function of rac, a ras-related GTP-binding protein, was investigated in fibroblasts by microinjection. In confluent serum-starved Swiss 3T3 cells, rac1 rapidly stimulated actin filament accumulation at the plasma Membrane, forming Membrane ruffles. Several growth factors and activated H-ras also induced Membrane Ruffling, and this response was prevented by a dominant inhibitory mutant rac protein, N17rac1. This suggests that endogenous rac proteins are required for growth factor-induced Membrane Ruffling. In addition to Membrane Ruffling, a later response to both rac1 microinjection and some growth factors was the formation of actin stress fibers, a process requiring endogenous rho proteins. Using N17rac1 we have shown that these growth factors act through rac to stimulate this rho-dependent response. We propose that rac and rho are essential components of signal transduction pathways linking growth factors to the organization of polymerized actin.
Lena Claessonwelsh - One of the best experts on this subject based on the ideXlab platform.
-
activation of phosphoinositide 3 kinase is required for pdgf stimulated Membrane Ruffling
1994Co-Authors: Stefan Wennstrom, Lena Claessonwelsh, Kazuyoshi Yonezawa, Phillip T Hawkins, Frank T Cooke, Kenta Hara, Masato Kasuga, T R Jackson, Len R StephensAbstract:Abstract Background: There is substantial evidence that phosphoinositide 3-kinase (PI 3-kinase) is a critical component of signalling pathways used by the cell-surface receptors for a variety of mammalian growth factors and other hormones. The physiological product of this enzyme is a highly polar Membrane lipid called phosphatidylinositol (3,4,5)-trisphosphate This lipid has been postulated to act as a second-messenger in cells but its putative targets are still unknown. Results A particular rearrangement of actin filaments, which results in Membrane Ruffling, is elicited by the activation of PDGF β -receptors expressed in cultured porcine aortic endothelial cells. We have found that this consequence of PDGF β -receptor activation is inhibited by three independent manipulations of PI 3-kinase activity: firstly, by the deletion of tyrosine residues in the PDGF β -receptor to which PI 3-kinase binds; secondly, by the overexpression of a mutant 85 kD PI 3-kinase regulatory subunit to which the catalytic kinase subunit cannot bind; and thirdly, by the addition of the fungal metabolite wortmannin, which is a potent inhibitor of the catalytic activity of PI 3-kinase. Conclusion These results argue strongly that phosphatidylinositol (3,4,5)-trisphosphate synthesis is required for growth-factor-stimulated Membrane Ruffling in porcine aortic endothelial cells, and suggest that synthesis of this lipid may be part of a signalling pathway leading to direct or indirect activation of the small GTP-binding protein Rac.
-
Membrane Ruffling and chemotaxis transduced by the pdgf beta receptor require the binding site for phosphatidylinositol 3 kinase
1994Co-Authors: Stefan Wennstrom, Annkristin Arvidsson, Seijiro Mori, Agneta Siegbahn, Carl-henrik Heldin, Koutaro Yokote, Lena ClaessonwelshAbstract:Membrane Ruffling and chemotaxis transduced by the PDGF beta-receptor require the binding site for phosphatidylinositol 3' kinase.
-
transduction of circular Membrane Ruffling by the platelet derived growth factor beta receptor is dependent on its kinase insert
1992Co-Authors: Annkristin Arvidsson, Carl-henrik Heldin, Lena ClaessonwelshAbstract:Abstrad The platelet-derived growth fador (PDGF) a- and $receptors both mediate a mitogenic response, but only the fl-receptor mediates circular adin reorganization and chemotaxis. The tyrosine kinase domains of the receptors contain noncatalytic inserts of about 100 residues. In order to determine the role of these domains in the differential signaling of the two receptors, we construded chimeric PDGF receptors and expressed the construds in porcine aortic endothelial cells. The chimeric receptors were similar to the wildtype receptors in their ability to induce mitogenicity in response to ligand. Examination of receptor-associated substrates by in vitro kinase assays revealed that phosphoproteins of 72 and 110 kilodaltons were associated with the kinase insert of the a-receptor, whereas a phosphoprotein of 130 kilodaltons was associated with the kinase insert of the �9-receptor. Adin reorganization in the form of circular Membrane Ruffling was seen after ligand stimulation of the $receptor and the a-receptor containing the fl-receptor kinase insert but not after stimulation of the a-receptor or the fl-receptor containing the a-receptor kinase insert. These data indicate that the PDGF fl-receptor kinase insert has an essential fundion in the signal transdudion pathway leading to circular Membrane Ruffling.
Alan Hall - One of the best experts on this subject based on the ideXlab platform.
-
salmonella typhimurium induces Membrane Ruffling by a growth factor receptor independent mechanism
1993Co-Authors: Bradley D Jones, Hugh Paterson, Alan Hall, Stanley FalkowAbstract:Abstract Invasive Salmonella typhimurium induces dramatic actin rearrangements on the Membrane surface of mammalian cells as part of its entry mechanism. These changes, which are best characterized as membranous ruffles, closely resemble the Membrane changes that occur when a growth factor binds to its receptor. Recently, inhibition of the function of the small GTPases rac and rho in quiescent serum-starved fibroblasts was demonstrated to abolish growth factor-mediated Ruffling and stress-fiber formation, respectively. In addition, actin changes induced by the oncogene ras were also shown to be regulated by rac and rho. Because Salmonella-induced actin rearrangements resemble those caused by growth factors, we investigated whether ras, rho, or rac regulates the Membrane Ruffling elicited by S. typhimurium. Surprisingly, inhibition of the functions of these GTPases had no effect on the ability of invasive S. typhimurium to induce Membrane ruffles on a variety of tissue culture cells including Madin-Darby canine kidney cells, Swiss 3T3 fibroblasts, and Hep-2 cells. These results led us to examine the interactions of S. typhimurium with Henle-407 intestinal cells, which lack epidermal growth factor receptor on their Membrane surface. We found no difference in the ability of invasive S. typhimurium to induce Membrane Ruffling and to enter Henle-407 cells with or without the epidermal growth factor receptor on the Membrane surface. We, therefore, conclude that invasive S. typhimurium induces Membrane Ruffling and its own internalization by a rac-independent, growth factor-receptor-independent signaling pathway.
-
the small gtp binding protein rac regulates growth factor induced Membrane Ruffling
1992Co-Authors: Anne J Ridley, Hugh Paterson, Caroline L Johnston, Dagmar Diekmann, Alan HallAbstract:The function of rac, a ras-related GTP-binding protein, was investigated in fibroblasts by microinjection. In confluent serum-starved Swiss 3T3 cells, rac1 rapidly stimulated actin filament accumulation at the plasma Membrane, forming Membrane ruffles. Several growth factors and activated H-ras also induced Membrane Ruffling, and this response was prevented by a dominant inhibitory mutant rac protein, N17rac1. This suggests that endogenous rac proteins are required for growth factor-induced Membrane Ruffling. In addition to Membrane Ruffling, a later response to both rac1 microinjection and some growth factors was the formation of actin stress fibers, a process requiring endogenous rho proteins. Using N17rac1 we have shown that these growth factors act through rac to stimulate this rho-dependent response. We propose that rac and rho are essential components of signal transduction pathways linking growth factors to the organization of polymerized actin.
Naoki Mochizuki - One of the best experts on this subject based on the ideXlab platform.
-
adaptor protein crk is required for ephrin b1 induced Membrane Ruffling and focal complex assembly of human aortic endothelial cells
2002Co-Authors: K Nagashima, Akira Endo, Hisakazu Ogita, Akiko Kawana, Akiko Yamagishi, Akira Kitabatake, Michiyuki Matsuda, Naoki MochizukiAbstract:Endothelial cell migration is an essential step in vasculogenesis and angiogenesis, in which receptor tyrosine kinases play a pivotal role. We investigated the mechanism by which ephrin-B1 promotes Membrane Ruffling in human aortic endothelial cells, because Membrane Ruffling heralds cell body migration. We especially focused on the role of Crk adaptor protein in EphB-mediated signaling. Using DsRed-tagged Crk and a fluorescent time-lapse microscope, we showed that Crk was recruited to the nascent focal complex after ephrin-B1 stimulation. Furthermore, we found that p130Cas, but not paxillin, recruited Crk to the nascent focal complex. The necessity of Crk in ephrin-B1–induced Membrane Ruffling was shown both by the overexpression of dominant negative Crk mutants and by the depletion of Crk by using RNA interference. Then, we examined the role of two major downstream molecules of Crk, Rac1 and Rap1. The dominant negative mutant of Rac1 completely inhibited ephrin-B1–induced Membrane Ruffling and focal complex assembly. In contrast, rap1GAPII, a negative regulator of Rap1, did not inhibit ephrin-B1–induced Membrane Ruffling. However, in rap1GAPII-expressing cells, ephrin-B1 did not induce Membrane spreading, probably due to instability of the focal complex. These results indicated that Crk plays a critical role in Rac1-induced Membrane Ruffling and Rap1-mediated nascent focal complex stabilization contributing to ephrin-B1–induced human aortic endothelial cells migration.
-
sphingosine 1 phosphate induces Membrane Ruffling and increases motility of human umbilical vein endothelial cells via vascular endothelial growth factor receptor and crkii
2002Co-Authors: Akira Endo, K Nagashima, Michiyuki Matsuda, Hitoshi Kurose, Seibu Mochizuki, Naoki MochizukiAbstract:Sphingosine 1-phosphate (S1P), a ligand for endothelial differentiation gene family proteins, is one of the most potent signal mediators released from activated platelets. Here, we report that S1P induces Membrane Ruffling of human umbilical vein endothelial cells (HUVECs) via the vascular endothelial growth factor receptor (VEGFR), Src family tyrosine kinase(s), and the CrkII adaptor protein. S1P induced prominent phosphorylation of CrkII in HUVECs, indicating that CrkII was involved in the S1P-induced signaling pathway. S1P-induced CrkII phosphorylation was blocked by pertussis toxin and overexpression of the carboxyl terminus of β-adrenergic receptor kinase, indicating that the βγ subunit of Gi was required for the phosphorylation. Notably, the S1P-induced CrkII phosphorylation was also abolished by inhibitors of VEGFR or Src family tyrosine kinases. By using Picchu, a real time monitoring protein for CrkII phosphorylation, we found that S1P induced rapid CrkII phosphorylation at Membrane ruffles. Finally, we observed that expression of a dominant negative mutant of CrkII inhibited the S1P-induced Membrane Ruffling and cell migration. These results delineated a novel S1P signaling pathway that involves sequential activation of Gi-coupled receptor(s), VEGFR, Src family tyrosine kinase(s), and the CrkII adaptor protein, and which is responsible for both the induction of Membrane Ruffling and the increase in cell motility.
-
swap 70 is a guanine nucleotide exchange factor that mediates signalling of Membrane Ruffling
2002Co-Authors: Masahiro Shinohara, Yoh Terada, Akihiro Iwamatsu, Azusa Shinohara, Naoki Mochizuki, Maiko Higuchi, Yukiko Gotoh, Sayoko Ihara, Satoshi NagataAbstract:Phosphoinositide-3-OH kinase (PI(3)K), activated through growth factor stimulation, generates a lipid second messenger, phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). PtdIns(3,4,5)P3 is instrumental in signalling pathways that trigger cell activation, cytoskeletal rearrangement, survival and other reactions. However, some targets of PtdIns(3,4,5)P3 are yet to be discovered. We demonstrate that SWAP-70, a unique signalling protein, specifically binds PtdIns(3,4,5)P3. On stimulation by growth factors, cytoplasmic SWAP-70, which is dependent on PI(3)K but independent of Ras, moved to cell Membrane rearrangements known as ruffles. However, mutant SWAP-70 lacking the ability to bind PtdIns(3,4,5)P3 blocked Membrane Ruffling induced by epidermal growth factor or platelet-derived growth factor. SWAP-70 shows low homology with Rac-guanine nucleotide exchange factors (GEFs), and catalyses PtdIns(3,4,5)P3-dependent guanine nucleotide exchange to Rac. SWAP-70-deficient fibroblasts showed impaired Membrane Ruffling after stimulation with epidermal growth factor, and failed to activate Rac fully. We conclude that SWAP-70 is a new type of Rac-GEF which, independently of Ras, transduces signals from tyrosine kinase receptors to Rac.
Kazuyoshi Yonezawa - One of the best experts on this subject based on the ideXlab platform.
-
phosphoinositide 3 kinase as an upstream regulator of the small gtp binding protein rac in the insulin signaling of Membrane Ruffling
1995Co-Authors: Ko Kotani, Kazuyoshi Yonezawa, Kenshiro Hara, M KasugaAbstract:Abstract Membrane Ruffling and the closely linked response of fluid-phase pinocytosis were investigated in Chinese hamster ovary cells that stably overexpress the human insulin receptor and a mutant 85-kDa subunit of phosphoinositide (PI) 3-kinase (Δp85) that lacks a binding site for the catalytic 110-kDa subunit of this enzyme. Both Membrane Ruffling and pinocytosis induced by insulin were markedly impaired in these cells. Microinjection of Rac, a Ras-related small GTP-binding protein, induced Membrane Ruffling in human epidermoid carcinoma KB cells, and this effect of Rac was not blocked by coinjection of Δp85 or by exposure of cells to wortmannin, a specific Pl 3-kinase inhibitor. These results suggest that PI 3-kinase is essential not only for insulin-stimulated Membrane Ruffling but also for pinocytosis, and that PI 3-kinase possibly functions upstream of Rac in the signal transduction pathway.
-
involvement of phosphoinositide 3 kinase in insulin or igf 1 induced Membrane Ruffling
1994Co-Authors: Ko Kotani, Kazuyoshi Yonezawa, Kenshiro Hara, H Ueda, Yukari Kitamura, Hiroshi Sakaue, A Ando, A Chavanieu, B Calas, Florin GrigorescuAbstract:Insulin, IGF-1 or EGF induce Membrane Ruffling through their respective tyrosine kinase receptors. To elucidate the molecular link between receptor activation and Membrane Ruffling, we microinjected phosphorylated peptides containing YMXM motifs or a mutant 85 kDa subunit of phosphoinositide (PI) 3-kinase (delta p85) which lacks a binding site for the catalytic 110 kDa subunit of PI 3-kinase into the cytoplasm of human epidermoid carcinoma KB cells. Both inhibited the association of insulin receptor substrate-1 (IRS-1) with PI 3-kinase in a cell-free system and also inhibited insulin- or IGF-1-induced, but not EGF-induced, Membrane Ruffling in KB cells. Microinjection of nonphosphorylated analogues, phosphorylated peptides containing the EYYE motif or wild-type 85 kDa subunit (Wp85), all of which did not inhibit the association of IRS-1 with PI 3-kinase in a cell-free system, did not inhibit Membrane Ruffling in KB cells. In addition, wortmannin, an inhibitor of PI 3-kinase activity, inhibited insulin- or IGF-1-induced Membrane Ruffling. These results suggest that the association of IRS-1 with PI 3-kinase followed by the activation of PI 3-kinase are required for insulin- or IGF-1-induced, but not for EGF-induced, Membrane Ruffling.
-
activation of phosphoinositide 3 kinase is required for pdgf stimulated Membrane Ruffling
1994Co-Authors: Stefan Wennstrom, Lena Claessonwelsh, Kazuyoshi Yonezawa, Phillip T Hawkins, Frank T Cooke, Kenta Hara, Masato Kasuga, T R Jackson, Len R StephensAbstract:Abstract Background: There is substantial evidence that phosphoinositide 3-kinase (PI 3-kinase) is a critical component of signalling pathways used by the cell-surface receptors for a variety of mammalian growth factors and other hormones. The physiological product of this enzyme is a highly polar Membrane lipid called phosphatidylinositol (3,4,5)-trisphosphate This lipid has been postulated to act as a second-messenger in cells but its putative targets are still unknown. Results A particular rearrangement of actin filaments, which results in Membrane Ruffling, is elicited by the activation of PDGF β -receptors expressed in cultured porcine aortic endothelial cells. We have found that this consequence of PDGF β -receptor activation is inhibited by three independent manipulations of PI 3-kinase activity: firstly, by the deletion of tyrosine residues in the PDGF β -receptor to which PI 3-kinase binds; secondly, by the overexpression of a mutant 85 kD PI 3-kinase regulatory subunit to which the catalytic kinase subunit cannot bind; and thirdly, by the addition of the fungal metabolite wortmannin, which is a potent inhibitor of the catalytic activity of PI 3-kinase. Conclusion These results argue strongly that phosphatidylinositol (3,4,5)-trisphosphate synthesis is required for growth-factor-stimulated Membrane Ruffling in porcine aortic endothelial cells, and suggest that synthesis of this lipid may be part of a signalling pathway leading to direct or indirect activation of the small GTP-binding protein Rac.