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Träfflista för sökning "WFRF:(Aspenström Pontus) "

Sökning: WFRF:(Aspenström Pontus)

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1.
  • Aspenström-Fagerlund, Bitte, et al. (författare)
  • Oleic acid and docosahexaenoic acid cause an increase in the paracellular absorption of hydrophilic compounds in an experimental model of human absorptive enterocytes
  • 2007
  • Ingår i: Toxicology. - : Elsevier BV. - 0300-483X .- 1879-3185. ; 237:1-3, s. 12-23
  • Tidskriftsartikel (refereegranskat)abstract
    • Surface active compounds present in food possibly have the ability to enhance the absorption of water soluble toxic agents. Therefore, we investigated whether fatty acids such as oleic acid and docosahexaenoic acid (DHA), both commonly present in food, negatively affect the integrity of tight junctions (TJ) in the intestinal epithelium and thereby increase the absorption of poorly absorbed hydrophilic substances. Caco-2 cells, which are derived from human absorptive enterocytes, were grown on permeable filters for 20-25 days. Differentiated cell monolayers were apically exposed for 90min to mannitol in emulsions of oleic acid (5, 15 or 30mM) or DHA (5, 15 or 30mM) in an experimental medium with or without Ca(2+) and Mg(2+). Absorption of (14)C-mannitol increased and trans-epithelial electrical resistance (TEER) decreased in cell monolayers exposed to oleic acid and DHA, compared to controls. Cytotoxicity, measured as leakage of LDH, was higher in groups exposed to 30mM oleic acid and all concentrations of DHA. Morphology of the cell monolayers was studied by using fluorescence microscopy. Exposure of cell monolayers to 5mM DHA for 90min resulted in a profound alteration of the cell-cell contacts as detected by staining the cells for beta-catenin. Oleic acid (30mM) treatment also induced dissolution of the cell-cell contacts but the effect was not as pronounced as with DHA. Cell monolayers were also exposed for 180min to 250nM cadmium (Cd) in emulsions of oleic acid (5 or 30mM) or DHA (1 or 5mM), in an experimental medium with Ca(2+) and Mg(2+). Retention of Cd in Caco-2 cells was higher after exposure to 5mM oleic acid but lower after exposure to 30mM oleic acid and DHA. Absorption of Cd through the monolayers increased after DHA exposure but not after exposure to oleic acid. Our results indicate that fatty acids may compromise the integrity of the intestinal epithelium and that certain lipids in food may enhance the paracellular absorption of poorly absorbed hydrophilic substances.
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2.
  • Aase, Karin, et al. (författare)
  • Angiomotin regulates endothelial cell migration during embryonic angiogenesis
  • 2007
  • Ingår i: Genes & Development. - : Cold Spring Harbor Laboratory. - 0890-9369 .- 1549-5477. ; 21:16, s. 2055-2068
  • Tidskriftsartikel (refereegranskat)abstract
    • The development of the embryonic vascular system into a highly ordered network requires precise control over the migration and branching of endothelial cells (ECs). We have previously identified angiomotin (Amot) as a receptor for the angiogenesis inhibitor angiostatin. Furthermore, DNA vaccination targeting Amot inhibits angiogenesis and tumor growth. However, little is known regarding the role of Amot in physiological angiogenesis. We therefore investigated the role of Amot in embryonic neovascularization during zebrafish and mouse embryogenesis. Here we report that knockdown of Amot in zebrafish reduced the number of filopodia of endothelial tip cells and severely impaired the migration of intersegmental vessels. We further show that 75% of Amot knockout mice die between embryonic day 11 (E11) and E11.5 and exhibit severe vascular insufficiency in the intersomitic region as well as dilated vessels in the brain. Furthermore, using ECs differentiated from embryonic stem (ES) cells, we demonstrate that Amot-deficient cells have intact response to vascular endothelial growth factor (VEGF) in regard to differentiation and proliferation. However, the chemotactic response to VEGF was abolished in Amot-deficient cells. We provide evidence that Amot is important for endothelial polarization during migration and that Amot controls Rac1 activity in endothelial and epithelial cells. Our data demonstrate a critical role for Amot during vascular patterning and endothelial polarization.
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3.
  • Aspenström, Pontus (författare)
  • Activated Rho GTPases in Cancer-The Beginning of a New Paradigm
  • 2018
  • Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 19:12
  • Forskningsöversikt (refereegranskat)abstract
    • Involvement of Rho GTPases in cancer has been a matter of debate since the identification of the first members of this branch of the Ras superfamily of small GTPases. The Rho GTPases were ascribed important roles in the cell, although these were restricted to regulation of cytoskeletal dynamics, cell morphogenesis, and cell locomotion, with initially no clear indications of direct involvement in cancer progression. This paradigm has been challenged by numerous observations that Rho-regulated pathways are often dysregulated in cancers. More recently, identification of point mutants in the Rho GTPases Rac1, RhoA, and Cdc42 in human tumors has finally given rise to a new paradigm, and we can now state with confidence that Rho GTPases serve as oncogenes in several human cancers. This article provides an expose of current knowledge of the roles of activated Rho GTPases in cancers.
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4.
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5.
  • Aspenström, Pontus (författare)
  • Integration of signalling pathways regulated by small GTPases and calcium
  • 2004
  • Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434. ; 1742:1-3, s. 51-58
  • Forskningsöversikt (refereegranskat)abstract
    • The Ras superfamily of small GTPases constitutes a large group of structurally and functionally related proteins. They function as signalling switches in numerous signalling cascades in the cell. During the recent years, an increased awareness of a communication between signalling systems employing Ras-like GTPases and signalling systems employing calcium has emerged. For instance, the intensity of the activation of Ras-like GTPases is regulated by calcium-dependent mechanisms, acting on proteins that facilitate the activation or inactivation of the small GTPases. Other Ras-like GTPases have a direct influence on calcium signalling by regulating the activity of certain calcium channels. In addition, several small GTPases collaborate with calcium signalling in regulating cellular processes, such as cell adhesion, cell migration and exocytosis.
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6.
  • Aspenström, Pontus (författare)
  • Miro GTPases at the Crossroads of Cytoskeletal Dynamics and Mitochondrial Trafficking
  • 2024
  • Ingår i: Cells. - : MDPI. - 2073-4409. ; 13:7
  • Forskningsöversikt (refereegranskat)abstract
    • Miro GTPases are key components in the machinery responsible for transporting mitochondria and peroxisomes along microtubules, and also play important roles in regulating calcium homeostasis and organizing contact sites between mitochondria and the endoplasmic reticulum. Moreover, Miro GTPases have been shown to interact with proteins that actively regulate cytoskeletal organization and dynamics, suggesting that these GTPases participate in organizing cytoskeletal functions and organelle transport. Derailed mitochondrial transport is associated with neuropathological conditions such as Parkinson's and Alzheimer's diseases. This review explores our recent understanding of the diverse roles of Miro GTPases under cytoskeletal control, both under normal conditions and during the course of human diseases such as neuropathological disorders.
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7.
  • Aspenström, Pontus, et al. (författare)
  • Pombe Cdc15 homology proteins : regulators of membrane dynamics and the actin cytoskeleton
  • 2006
  • Ingår i: TIBS -Trends in Biochemical Sciences. Regular ed.. - : Elsevier BV. - 0968-0004 .- 1362-4326. ; 31:12, s. 670-679
  • Forskningsöversikt (refereegranskat)abstract
    • Pombe Cdc15 homology (PCH) proteins have emerged in many species as important coordinators of signalling pathways that regulate actomyosin assembly and membrane dynamics. For example, the prototype PCH protein, Cdc15p of Schizosaccharomyces pombe, has a role in assembly of the contractile ring, which is needed to separate dividing cells. Recently, mammalian PCH proteins have been found to bind phospholipids and to participate in membrane deformation. These findings suggest that PCH proteins are crucial linkers of membrane dynamics and actin polymerization, for example, during the internalization of transmembrane receptors. Intriguingly, some members of the PCH protein family are mutated in neurodegenerative and inflammatory diseases, which has implications for the identification of cures for such disorders.
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8.
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9.
  • Aspenström, Pontus, et al. (författare)
  • Rho GTPases have diverse effects on the organization of the actin filament system
  • 2004
  • Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 377:Pt 2, s. 327-337
  • Tidskriftsartikel (refereegranskat)abstract
    • The Rho GTPases are related to the Ras proto-oncogenes and consist of 22 family members. These proteins have important roles in regulating the organization of the actin filament system, and thereby the morphogenesis of vertebrate cells as well as their ability to migrate. In an effort to compare the effects of all members of the Rho GTPase family, active Rho GTPases were transfected into porcine aortic endothelial cells and the effects on the actin filament system were monitored. Cdc42, TCL (TC10-like), Rac1-Rac3 and RhoG induced the formation of lamellipodia, whereas Cdc42, Rac1 and Rac2 also induced the formation of thick bundles of actin filaments. In contrast, transfection with TC10 or Chp resulted in the formation of focal adhesion-like structures, whereas Wrch-1 induced long and thin filopodia. Transfection with RhoA, RhoB or RhoC induced the assembly of stress fibres, whereas Rnd1-Rnd3 resulted in the loss of stress fibres, but this effect was associated with the formation of actin- and ezrin-containing dorsal microvilli. Cells expressing RhoD and Rif had extremely long and flexible filopodia. None of the RhoBTB or Miro GTPases had any major influence on the organization of the actin filament system; instead, RhoBTB1 and RhoBTB2 were present in vesicular structures, and Miro-1 and Miro-2 were present in mitochondria. Collectively, the data obtained in this study to some extent confirm earlier observations, but also allow the identification of previously undetected roles of the different members of the Rho GTPases.
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10.
  • Aspenström, Pontus (författare)
  • Roles of F-BAR/PCH Proteins in the Regulation of Membrane Dynamics and Actin Reorganization
  • 2009
  • Ingår i: International Review of Cell and Molecular Biology. ; 272, s. 1-31
  • Forskningsöversikt (refereegranskat)abstract
    • The Pombe Cdc15 Homology (PCH) proteins have emerged in many species as important coordinators of signaling pathways that regulate actomyosin assembly and membrane dynamics. The hallmark of the PCH proteins is the presence of a Fes/ClP4 homology-Bin/Amphiphysin/Rvsp (F-BAR) domain; therefore they are commonly referred to as F-BAR proteins. The prototype F-BAR protein, Cdc15p of Schizosaccharomyces pombe, has a role in the formation of the contractile actomyosin ring during cytokinesis. Vertebrate F-BAR proteins have an established role in binding phospholipids and they participate in membrane deformations, for instance, during the internalization of transmembrane receptors. This way the F-BAR proteins will function as linkers between the actin polymerization apparatus and the machinery regulating membrane dynamics. Interestingly, some members of the F-BAR proteins are implicated in inflammatory or neurodegenerative disorders and the observations can be expected to have clinical implications for the treatment of the diseases.
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11.
  • Aspenström, Pontus, et al. (författare)
  • Taking Rho GTPases to the next level : the cellular functions of atypical Rho GTPases
  • 2007
  • Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 313:17, s. 3673-3679
  • Forskningsöversikt (refereegranskat)abstract
    • The Rho GTPases are influential regulators of signalling pathways that control vital cellular processes such as cytoskeletal dynamics, gene transcription, cell cycle progression and cell transformation. A vast majority of the studies involving Rho GTPases have been focused to the famous triad, Cdc42, Rac1 and RhoA, but this protein family actually harbours 20 members. Recently, the less known Rho GTPases have received increased attention. Many of the less studied Rho GTPases have structural, as well as, functional features which makes it pertinent to classify them as atypical Rho GTPases. This review article will focus on the critical aspects of the atypical Rho GTPases, RhoH, Wrch-1, Chp and RhoBTB. These proteins are involved in a broad spectre of biological processes, such as cytoskeletal dynamics, T-cell signalling and protein ubiquitinylation. We will also discuss the roles of atypical Rho GTPases as oncogenes or tumour suppressors, as well as their potential involvement in human diseases.
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12.
  • Aspenström, Pontus, et al. (författare)
  • The diaphanous-related formin DAAM1 collaborates with the Rho GTPases RhoA and Cdc42, CIP4 and Src in regulating cell morphogenesis and actin dynamics
  • 2006
  • Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 312:12, s. 2180-2194
  • Tidskriftsartikel (refereegranskat)abstract
    • Binding partners for the Cdc42 effector CIP4 were identified by the yeast two-hybrid system, as well as by testing potential CIP4-binding proteins in coimmunoprecipitation experiments. One of the CIP4-binding proteins, DAAM1, was characterised in more detail. DAAM1 is a ubiquitously expressed member of the mammalian diaphanous-related formins, which include proteins such as mDia1 and mDia2. DAAM1 was shown to bind to the SH3 domain of CIP4 in vivo. Ectopically expressed DAAM1 localised in dotted pattern at the dorsal side of transfected cells and the protein was accumulated in the proximity to the microtubule organising centre. Moreover, ectopic expression of DAAM1 induced a marked alteration of the cell morphology, seen as rounding up of the cells, the formation of branched protrusions as well as a reduction of stress-fibres in the transfected cells. Coimmunoprecipitation experiments demonstrated that DAAM1 bound to RhoA and Cdc42 in a GTP-dependent manner. Moreover, DAAM1 was found to interact and collaborate with the non-receptor tyrosine kinase Src in the formation of branched protrusions. Taken together, our data indicate that DAAM1 communicates with Rho GTPases, CIP4 and Src in the regulation of the signalling pathways that co-ordinate the dynamics of the actin filament system.
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13.
  • Aspenström, Pontus (författare)
  • The Intrinsic GDP/GTP Exchange Activities of Cdc42 and Rac1 Are A Critical Determinants for Their Specific Effects on Mobilization of the Actin Filament System
  • 2019
  • Ingår i: Cells. - : MDPI AG. - 2073-4409. ; 8:7
  • Tidskriftsartikel (refereegranskat)abstract
    • The Rho GTPases comprise a subfamily of the Ras superfamily of small GTPases. Their importance in regulation of cell morphology and cell migration is well characterized. According to the prevailing paradigm, Cdc42 regulates the formation of filopodia, Rac1 regulates the formation of lamellipodia, and RhoA triggers the assembly of focal adhesions. However, this scheme is clearly an oversimplification, as the Rho subfamily encompasses 20 members with diverse effects on a number of vital cellular processes, including cytoskeletal dynamics and cell proliferation, migration, and invasion. This article highlights the importance of the catalytic activities of the classical Rho GTPases Cdc42 and Rac1, in terms of their specific effects on the dynamic reorganization of the actin filament system. GTPase-deficient mutants of Cdc42 and Rac1 trigger the formation of broad lamellipodia and stress fibers, and fast-cycling mutations trigger filopodia formation and stress fiber dissolution. The filopodia response requires the involvement of the formin family of actin nucleation promotors. In contrast, the formation of broad lamellipodia induced by GTPase-deficient Cdc42 and Rac1 is mediated through Arp2/3-dependent actin nucleation.
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14.
  • Aspenström, Pontus (författare)
  • The mammalian verprolin homologue WIRE participates in receptor-mediated endocytosis and regulation of the actin filament system by distinct mechanisms
  • 2004
  • Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 298:2, s. 485-498
  • Tidskriftsartikel (refereegranskat)abstract
    • The mammalian verprolin family consists of three family members: WIP, WIRE and CR16. WIRE was recently found to bind to WASP and N-WASP and to have roles in regulating actin dynamics downstream of the platelet-derived growth factor beta-receptor. In the current study, the WASP-binding domain of WIRE was identified, with the core of the binding motif encompassing amino acid residues 408-412. A stretch of aromatic amino acid residues close to the core motif also participates in WASP binding. Amino acid substitutions in each of these motifs abrogated WASP binding, suggesting that both motifs are involved in the binding of WIRE to WASP. Interestingly, WIRE mutants unable to bind WASP were still able to induce a reorganisation of the actin filament system, indicating that WASP did not participate in the signalling pathway that link WIRE to actin dynamics. In cells ectopically expressing WIRE, the endocytosis of the platelet-derived growth factor beta-receptor was drastically reduced. However, in contrast to the effect on the actin filament system, the WIRE-induced ablation of the receptor endocytosis required an intact WASP-binding domain. Moreover, WIRE was more efficient than WIP in inhibiting the receptor endocytosis, implicating that these two mammalian verprolins have distinct roles in mammalian cells.
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15.
  • Aspenström, Pontus (författare)
  • The Role of Fast-Cycling Atypical RHO GTPases in Cancer
  • 2022
  • Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 14:8
  • Forskningsöversikt (refereegranskat)abstract
    • For many years, cancer-associated mutations in RHO GTPases were not identified and observations suggesting roles for RHO GTPases in cancer were sparse. Instead, RHO GTPases were considered primarily to regulate cell morphology and cell migration, processes that rely on the dynamic behavior of the cytoskeleton. This notion is in contrast to the RAS proteins, which are famous oncogenes and found to be mutated at high incidence in human cancers. Recent advancements in the tools for large-scale genome analysis have resulted in a paradigm shift and RHO GTPases are today found altered in many cancer types. This review article deals with the recent views on the roles of RHO GTPases in cancer, with a focus on the so-called fast-cycling RHO GTPases. The RHO GTPases comprise a subfamily within the RAS superfamily of small GTP-hydrolyzing enzymes and have primarily been ascribed roles in regulation of cytoskeletal dynamics in eukaryotic cells. An oncogenic role for the RHO GTPases has been disregarded, as no activating point mutations were found for genes encoding RHO GTPases. Instead, dysregulated expression of RHO GTPases and their regulators have been identified in cancer, often in the context of increased tumor cell migration and invasion. In the new landscape of cancer genomics, activating point mutations in members of the RHO GTPases have been identified, in particular in RAC1, RHOA, and CDC42, which has suggested that RHO GTPases can indeed serve as oncogenes in certain cancer types. This review describes the current knowledge of these cancer-associated mutant RHO GTPases, with a focus on how their altered kinetics can contribute to cancer progression.
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16.
  • Aspenström, Pontus (författare)
  • The verprolin family of proteins : Regulators of cell morphogenesis and endocytosis
  • 2005
  • Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 579:24, s. 5253-5259
  • Forskningsöversikt (refereegranskat)abstract
    • The verprolin family of proteins, WIP, CR16 and WIRE/WICH, has emerged as critical regulators of cytoskeletal organisation in vertebrate cells. The founding father of the family, verprolin, was originally identified in budding yeast and later shown to be needed for actin polymerisation during polarised growth and during endocytosis. The vertebrate verprolins regulate actin dynamics either by binding directly to actin, by binding the WASP family of proteins or by binding to other actin regulating proteins. Interestingly, also the vertebrate verprolins have been implicated in endocytosis, demonstrating that most of the functional modules in this fascinating group of proteins have been conserved from yeast to man.
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17.
  • Aspenström, Pontus (författare)
  • The verprolins as regulators of actin dynamics.
  • 2006
  • Ingår i: Actin-monomer-binding proteins.. - Austin, Texas : Landes Biosciences. - 9780387464053 ; , s. 97-106
  • Bokkapitel (refereegranskat)abstract
    • Verprolin is an actin-binding protein first identified in budding yeast Saccharomyces cerevisiae. The yeast verprolin is needed for actin polymerisation during polarised growth and during endocytosis. In vertebrate cells, three genes encoding Verprolin orthologues have been identified: WIP, CR16 and WIRE/WICH. The mammalian verprolins have been implicated in the regulation of actin dynamics either by binding directly to actin, by binding the WASP family of proteins or by binding to other actin regulating proteins. This review article will bring up to discussion the current understanding of the mechanisms underlying verprolin-dependent mobilisation of the actin filament system.
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18.
  • Aspenström, Pontus (författare)
  • The WASP-binding protein WIRE has a role in the regulation of the actin filament system downstream of the platelet-derived growth factor receptor
  • 2002
  • Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 279:1, s. 21-33
  • Tidskriftsartikel (refereegranskat)abstract
    • Activation of growth factor receptors, such as platelet-derived growth factor (PDGF) receptors, has a major impact on the motile behavior of vertebrate cells. The WASP family of proteins has been recognized as important regulators of actin polymerization via the activation of the Arp2/3 complex. The activity of the WASP proteins has, in turn, been shown to be governed by a number of associated proteins, including the WASP interacting protein (WIP). This report presents a novel WIP-like protein, WIRE (for WIP-related). WIRE was shown to bind to the WH1 domain of WASP and N-WASP. WIRE was localized to actin filaments in transiently transfected PAE/PDGFRbeta cells, and in cells simultaneously expressing WIRE and WASP, WIRE relocalized WASP to actin filaments, a relocalization that required direct interaction between the two proteins. In addition, WIRE was able to bind the PDGF receptor substrate Nckbeta. PDGF treatment of cells ectopically expressing WIRE resulted in formation of peripheral protrusions composed of filopodia and lamellipodia-like structures. In cells expressing both WIRE and WASP, PDGF treatment induced a translocation of WASP to the cell margin, an effect that required the presence of WIRE. Taken together, the data presented indicate that WIRE has a role in the WASP-mediated organization of the actin cytoskeleton and that WIRE is a potential link between the activated PDGF receptor and the actin polymerization machinery.
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19.
  • Berthold, Jessica, et al. (författare)
  • Characterization of RhoBTB-dependent Cul3 ubiquitin ligase complexes--evidence for an autoregulatory mechanism
  • 2008
  • Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 314:19, s. 3453-3465
  • Tidskriftsartikel (refereegranskat)abstract
    • RhoBTB proteins are atypical members of the Rho family of small GTPases. Two of the three RhoBTB proteins, RhoBTB1 and RhoBTB2, have been proposed as tumor suppressors and might function as adaptors of Cul3-dependent ubiquitin ligase complexes. Using yeast two-hybrid analysis and co-immunoprecipitation we show that all three RhoBTB proteins interact with Cul3. The interaction requires the N-terminal region of Cul3 and the first BTB domain of RhoBTB. RhoBTB3, the only RhoBTB with a prenylation motif, associates with vesicles that are frequently found in the vicinity of microtubules, suggesting a participation in some aspects of vesicle trafficking. We also show that RhoBTB2 and RhoBTB3 are capable of homo and heterodimerizing through the BTB domain region. The GTPase domain, which does not bind GTP, is able to interact with the BTB domain region, thus preventing proteasomal degradation of RhoBTB. This fits into a model in which an intramolecular interaction maintains RhoBTB in an inactive state, preventing the formation or the functionality of Cul3-dependent complexes. We also report a significantly decreased expression of RHOBTB and CUL3 genes in kidney and breast tumor samples and a very good correlation in the expression changes between RHOBTB and CUL3 that suggests that these genes are subject to a common inactivation mechanism in tumors.
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21.
  • Blom, Magdalena, et al. (författare)
  • The atypical Rho GTPase RhoD is a regulator of actin cytoskeleton dynamics and directed cell migration
  • 2017
  • Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 352:2, s. 255-264
  • Tidskriftsartikel (refereegranskat)abstract
    • RhoD belongs to the Rho GTPases, a protein family responsible for the regulation and organization of the actin cytoskeleton, and, consequently, many cellular processes like cell migration, cell division and vesicle trafficking. Here, we demonstrate that the actin cytoskeleton is dynamically regulated by increased or decreased protein levels of RhoD. Ectopic expression of RhoD has previously been shown to give an intertwined weave of actin filaments. We show that this RhoD-dependent effect is detected in several cell types and results in a less dynamic actin filament system. In contrast, RhoD depletion leads to increased actin filament-containing structures, such as cortical actin, stress fibers and edge ruffles. Moreover, vital cellular functions such as cell migration and proliferation are defective when RhoD is silenced. Taken together, we present data suggesting that RhoD is an important component in the control of actin dynamics and directed cell migration.
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22.
  • Chiara, Federica, et al. (författare)
  • A gain of function mutation in the activation loop of platelet-derived growth factor beta-receptor deregulates its kinase activity
  • 2004
  • Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 279:41, s. 42516-42527
  • Tidskriftsartikel (refereegranskat)abstract
    • The platelet-derived growth factor receptors (PDGFRs) are receptor tyrosine kinases implicated in multiple aspects of cell growth, differentiation, and survival. Recently, a gain of function mutation in the activation loop of the human PDGFRalpha has been found in patients with gastrointestinal stromal tumors. Here we show that a mutation in the corresponding codon in the activation loop of the murine PDGFRbeta, namely an exchange of asparagine for aspartic acid at amino acid position 849 (D849N), confers transforming characteristics to embryonic fibroblasts from mutant mice, generated by a knock-in strategy. By comparing the enzymatic properties of the wild-type versus the mutant receptor protein, we demonstrate that the D849N mutation lowers the threshold for kinase activation, causes a dramatic alteration in the pattern of tyrosine phosphorylation kinetics following ligand stimulation, and induces a ligand-independent phosphorylation of several tyrosine residues. These changes result in deregulated recruitment of specific signal transducers. The GTPase-activating protein for Ras (RasGAP), a negative regulator of the Ras mitogenic pathway, displayed a delayed binding to the mutant receptor. Moreover, we have observed enhanced ligand-independent ERK1/2 activation and an increased proliferation of mutant cells. The p85 regulatory subunit of the phosphatidylinositol 3 '-kinase was constitutively associated with the mutant receptor, and this ligand-independent activation of the phosphatidylinositol 3'-kinase pathway may explain the observed strong protection against apoptosis and increased motility in cellular wounding assays. Our findings support a model whereby an activating point mutation results in a deregulated PDGFRbeta with oncogenic predisposition.
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23.
  • Dib, Karim, et al. (författare)
  • Down-regulation of Rac activity during beta 2 integrin-mediated adhesion of human neutrophils
  • 2003
  • Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 278:26, s. 24181-24188
  • Tidskriftsartikel (refereegranskat)abstract
    • In human neutrophils, beta2 integrin engagement mediated a decrease in GTP-bound Rac1 and Rac2. Pretreatment of neutrophils with LY294002 or PP1 (inhibiting phosphatidylinositol 3-kinase (PI 3-kinase) and Src kinases, respectively) partly reversed the beta2 integrin-induced down-regulation of Rac activities. In contrast, beta2 integrins induced stimulation of Cdc42 that was independent of Src family members. The PI 3-kinase dependence of the beta2 integrin-mediated decrease in GTP-bound Rac could be explained by an enhanced Rac-GAP activity, since this activity was blocked by LY204002, whereas PP1 only had a minor effect. The fact that only Rac1 but not Rac2 (the dominating Rac) redistributed to the detergent-insoluble fraction and that it was independent of GTP loading excludes the possibility that down-regulation of Rac activities was due to depletion of GTP-bound Rac from the detergent-soluble fraction. The beta2 integrin-triggered relocalization of Rac1 to the cytoskeleton was enabled by a PI 3-kinase-induced dissociation of Rac1 from LyGDI. The dissociations of Rac1 and Rac2 from LyGDI also explained the PI 3-kinase-dependent translocations of Rac GTPases to the plasma membrane. However, these accumulations of Rac in the membrane, as well as that of p47phox and p67phox, were also regulated by Src tyrosine kinases. Inasmuch as Rac GTPases are part of the NADPH oxidase and the respiratory burst is elicited in neutrophils adherent by beta2 integrins, our results indicate that activation of the NADPH oxidase does not depend on the levels of Rac-GTP but instead requires a beta2 integrin-induced targeting of the Rac GTPases as well as p47phox and p67phox to the plasma membrane.
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24.
  • Edling, Charlotte, 1976- (författare)
  • Receptor tyrosine kinase c-Kit signalling in hematopoietic progenitor cells
  • 2006
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • The receptor tyrosine kinase (RTK) c-Kit is expressed in hematopoietic stem and progenitor cells, mast cells and in several non-hematopoietic tissues. In the hematopoietic system c-Kit and its ligand Steel Factor (SF, aka Stem Cell Factor) are critical for proliferation, survival and differentiation. Mutations in either receptor or ligand lead to lethal anaemia, hematopoietic stem cell defects, mast cell deficiency and a series of non-hematological defects. The aims of the studies included in this thesis are to describe the signalling pathways downstream c-Kit in hematopoietic stem/progenitor cells and to further analyse the role of c-Kit signalling in fundamental biological functions. To study c-Kit signalling in the hematopoietic system we have employed hematopoietic stem cell-like cell lines which share many properties with primary hematopoietic stem cells in vitro and in vivo, including surface markers, multipotentiality, capacity for self-renewal and long term repopulation. In paper I we demonstrate that upon SF activation the RTK c-Kit is autophosphorylated and downstream signalling mediators are transiently activated. Surprisingly we find that the c-Kit mediated activation of the MAPK pathway is dependent on the activation of phosphoinositide 3-kinase (PI3K) in hematopoietic progenitor cells and that differentiation of these progenitors to mast cells results in a signalling switch where Raf activation changes from PI3K dependent to PI3K independent. We here establish that PI3K activity is required for viability and proliferation of hematopoietic progenitor cells. In paper II we studied the conventional protein kinase C (cPKC) involvement in c-Kit signalling. We observe that the cPKCs can phosphorylate c-Kit on serine 746 and that this phosphorylation negatively regulates the activation of the receptor. We demonstrate that inhibition of this negative phosphorylation results in dramatically increased protein kinase B (PKB) activation and as a consequence inhibition of cPKCs rescues cells from starvation induced apoptosis. Moreover we exhibit that the cPKCs are necessary for full activation of extracellular signal-regulated kinase (Erk) and that impaired PKC activity leads to hampered proliferation. In paper III we demonstrate that in addition to the cPKCs also the novel PKC is required for Erk activation and proliferation. Furthermore we present results indicating that PKC negatively regulates differentiation of bone marrow. In conclusion, with the studies in this thesis we display details in the signalling pathways induced upon RTK c-Kit activation and we demonstrate that c-Kit has significant effects on hematopoietic cell-physiology.
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25.
  • Edlund, Sofia, et al. (författare)
  • Interaction between Smad7 and beta-catenin : importance for transforming growth factor beta-induced apoptosis
  • 2005
  • Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 25:4, s. 1475-1488
  • Tidskriftsartikel (refereegranskat)abstract
    • Members of the transforming growth factor beta (TGF-beta) and Wnt/wingless superfamilies regulate cell fate during development and tissue maintenance. Here we report that Smad7 interacts with beta-catenin and lymphoid enhancer binding factor 1/T-cell-specific factor (LEF1/TCF), transcriptional regulators in Wnt signaling, in a TGF-beta-dependent manner. Smad7 was found to be required for TGF-beta1-induced accumulation of beta-catenin and LEF1 in human prostate cancer (PC-3U) cells as well as in human keratinocytes (HaCaT cells). Moreover, when the endogenous Smad7 was repressed by specific small interfering RNA, TGF-beta-induced increase of activated p38, Akt phosphorylated on Ser473, glycogen synthase kinase 3beta phosphorylated on Ser9 was prevented, as well as the TGF-beta-induced association between beta-catenin and LEF1. Notably, the observed physical association of Smad7 and beta-catenin was found to be important for TGF-beta-induced apoptosis, since suppression of beta-catenin expression by small interfering RNA decreased the apoptotic response to TGF-beta.
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26.
  • Edlund, Sofia (författare)
  • Mechanisms for TGF-β-Mediated Regulation of the Actin Filament System and Apoptosis
  • 2003
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Transforming growth factor-β (TGF-β) is a member of a large superfamily of cytokines which participate in many different types of cellular processes, such as growth inhibition, cell migration, differentiation, cell adhesion, wound healing and immunosuppression. Alterations of TGF-β superfamily signalling results in several different disorders, including bone disease, vascular disease and cancer. The TGF-β signalling pathways involve several different proteins, such as the Smad proteins, which upon receptor activation are translocated to the nucleus, where they affect transcriptional responses. The actin cytoskeleton is an organised network of filaments with a highly dynamic structure, which is under a continuous reconstruction to control the morphology, survival, growth and motility of eukaryotic cells. The members of the family of small GTP-binding proteins have been shown to be important regulators of the actin cytoskeleton.TGF-β was found to induce short term as well as long term actin reorganisation in prostate cancer cells. The short term response included membrane ruffling, and required signalling by the small GTPases Cdc42 and Rho as well as, the involvement of the mitogen-activated protein kinases p38 (p38 MAPK). The long term response included formation of stress fibers and required a cooperation between Smad and Rho GTPase signalling pathways involving the Rho-associated coiled-coil-containing protein kinase 1 (ROCK1). The TGF-β-induced activation of Cdc42 was, furthermore, shown to require the inhibitory Smad7 and p38 MAP kinase, via a PI3K-dependent pathway. Mixed lineage kinase 3 (MLK3), a mediator downstream of Cdc42, was necessary for the Cdc42-dependent actin filament reorganisation.Apoptosis is an important and carefully regulated process in human development and disease, which allows the multicellular organisms to remove cells that are in excess or potentially dangerous. TGF-β family members can induce apoptosis in many different cell types, in the presence or absence of other growth factors. Smad7 had previously been shown to be necessary for TGF-β-induced apoptosis of epithelial cells. We could show that Smad7 is required for TGF-β-induced activation of the TGF-β activated kinase 1 (TAK1)-mitogen-activated protein kinase kinase 3 (MKK3)-p38 MAPK pathway, which subsequently leads to apoptosis in prostate cancer cells.Members of the lymphoid enhancer factor-1/T-cell factor (LEF1/TCF) family of transcription factors have, together with β-catenin, been shown to be nuclear effectors in the Wnt-signalling pathway. We investigated a possible cross-talk between the TGF-β and Wnt signalling pathways. We found that TGF-β, in a Smad7-dependent manner induced a nuclear accumulation of β-catenin and enhanced the transcriptional activity of β-catenin and the induction of the downstream target gene c-myc. Since β-catenin and c-Myc has been shown to promote apoptosis, our results suggests the possibility that β-catenin contributes to TGF-β-induced apoptosis
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27.
  • Edlund, Sofia, et al. (författare)
  • Smad7 is required for TGF-ß-induced activation of the small GTPase Cdc42
  • 2004
  • Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 117:Pt 9, s. 1835-1847
  • Tidskriftsartikel (refereegranskat)abstract
    • Transforming growth factor beta (TGF-beta) is a potent regulator of cell growth and differentiation in many cell types. The Smad signaling pathway constitutes a main signal transduction route downstream of TGF-beta receptors. The inhibitory Smads, Smad6 and Smad7, are considered to function as negative regulators of the TGF-beta/Smad signaling cascade. In a previous study, we found that TGF-beta induces rearrangements of the actin filament system in human prostate carcinoma cells and that this response requires the small GTPases Cdc42 and RhoA. On the basis of the current view on the function of Smad7 in TGF-beta signaling, we hypothesized that Smad7 would function as a negative regulator of the TGF-beta-induced activation of Cdc42 and RhoA, but instead we found that the reverse is the case; Smad7 is required for the TGF-beta-induced activation of Cdc42 and the concomitant reorganization of the actin filament system. These observations propose a novel role for Smad7 in TGF-beta-dependent activation of Rho GTPases.
  •  
28.
  • Edlund, Sofia, et al. (författare)
  • Transforming growth factor-beta-induced mobilization of actin cytoskeleton required signaling by small GTPases Cdc42 and RhoA
  • 2002
  • Ingår i: Molecular Biology of the Cell. - : The American Society for Cell Biology. - 1059-1524 .- 1939-4586. ; 13:3, s. 902-914
  • Tidskriftsartikel (refereegranskat)abstract
    • Transforming growth factor-beta (TGF-beta) is a potent regulator of cell growth and differentiation in many cell types. The Smad signaling pathway constitutes a main signal transduction route downstream of TGF-beta receptors. We studied TGF-beta-induced rearrangements of the actin filament system and found that TGF-beta 1 treatment of PC-3U human prostate carcinoma cells resulted in a rapid formation of lamellipodia. Interestingly, this response was shown to be independent of the Smad signaling pathway; instead, it required the activity of the Rho GTPases Cdc42 and RhoA, because ectopic expression of dominant negative mutant Cdc42 and RhoA abrogated the response. Long-term stimulation with TGF-beta 1 resulted in an assembly of stress fibers; this response required both signaling via Cdc42 and RhoA, and Smad proteins. A known downstream effector of Cdc42 is p38(MAPK); treatment of the cells with the p38(MAPK) inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(pyridyl)1H-imidazole (SB203580), as well as ectopic expression of a kinase-inactive p38(MAPK), abrogated the TGF-beta-induced actin reorganization. Moreover, treatment of cells with the inhibitors of the RhoA target-protein Rho-associated coiled-coil kinase (+)-R-trans-4-(aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide (Y-27632) and 1-5(-isoquinolinesulfonyl)homopiperazine (HA-1077), as well as ectopic expression of kinase-inactive Rho coiled-coil kinase-1, abrogated the TGF-beta 1-induced formation of stress fibers. Collectively, these data indicate that TGF-beta-induced membrane ruffles occur via Rho GTPase-dependent pathways, whereas long-term effects require cooperation between Smad and Rho GTPase signaling pathways.
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29.
  • Edlund, Sofia, et al. (författare)
  • Transforming growth factor-beta1-induced apoptosis of prostate cancer cells involves Smad7-dependent activation of p38 by TGF-beta-activated kinase 1 and mitogen-activated protein kinase kinase 3
  • 2003
  • Ingår i: Molecular Biology of the Cell. - : The American Society for Cell Biology. - 1059-1524 .- 1939-4586. ; 14:2, s. 529-544
  • Tidskriftsartikel (refereegranskat)abstract
    • The inhibitory Smad7, a direct target gene for transforming growth factor-beta (TGF-beta), mediates TGF-beta1-induced apoptosis in several cell types. Herein, we report that apoptosis of human prostate cancer PC-3U cells induced by TGF-beta1 or Smad7 overexpression is caused by a specific activation of the p38 mitogen-activated protein kinase pathway in a TGF-beta-activated kinase 1 (TAK1)- and mitogen-activated protein kinase kinase 3 (MKK3)-dependent manner. Expression of dominant negative p38, dominant negative MKK3, or incubation with the p38 selective inhibitor [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole], prevented TGF-beta1-induced apoptosis. The expression of Smad7 was required for TGF-beta-induced activation of MKK3 and p38 kinases, and endogenous Smad7 was found to interact with phosphorylated p38 in a ligand-dependent manner. Ectopic expression of wild-type TAK1 promoted TGF-beta1-induced phosphorylation of p38 and apoptosis, whereas dominant negative TAK1 reduced TGF-beta1-induced phosphorylation of p38 and apoptosis. Endogenous Smad7 was found to interact with TAK1, and TAK1, MKK3, and p38 were coimmunoprecipitated with Smad7 in transiently transfected COS1 cells. Moreover, ectopically expressed Smad7 enhanced the coimmunoprecipitation of HA-MKK3 and Flag-p38, supporting the notion that Smad7 may act as a scaffolding protein and facilitate TAK1- and MKK3-mediated activation of p38.
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30.
  • Eriksson, Therese, et al. (författare)
  • Characterisation of the role of Vrp1 in cell fusion during the development of visceral muscle of Drosophila melanogaster
  • 2010
  • Ingår i: BMC Developmental Biology. - : BioMed Central. - 1471-213X. ; 10, s. 86-
  • Tidskriftsartikel (refereegranskat)abstract
    • BackgroundIn Drosophila muscle cell fusion takes place both during the formation of the somatic mesoderm and the visceral mesoderm, giving rise to the skeletal muscles and the gut musculature respectively. The core process of myoblast fusion is believed to be similar for both organs. The actin cytoskeleton regulator Verprolin acts by binding to WASP, which in turn binds to the Arp2/3 complex and thus activates actin polymerization. While Verprolin has been shown to be important for somatic muscle cell fusion, the function of this protein in visceral muscle fusion has not been determined.ResultsVerprolin is specifically expressed in the fusion competent myoblasts of the visceral mesoderm, suggesting a role in visceral mesoderm fusion. We here describe a novel Verprolin mutant allele which displays subtle visceral mesoderm fusion defects in the form of mislocalization of the immunoglobulin superfamily molecule Duf/Kirre, which is required on the myoblast cell surface to facilitate attachment between cells that are about to fuse, indicating a function for Verprolin in visceral mesoderm fusion. We further show that Verprolin mutant cells are capable of both migrating and fusing and that the WASP-binding domain of Verprolin is required for rescue of the Verprolin mutant phenotype.ConclusionsVerprolin is expressed in the visceral mesoderm and plays a role in visceral muscle fusion as shown by mislocalization of Duf/Kirre in the Verprolin mutant, however it is not absolutely required for myoblast fusion in either the visceral or the somatic mesoderm.
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31.
  • Fransson, Åsa, et al. (författare)
  • Atypical Rho GTPases have roles in mitochondrial homeostasis and apoptosis
  • 2003
  • Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 278:8, s. 6495-6502
  • Tidskriftsartikel (refereegranskat)abstract
    • The human genomic sequencing effort has revealed the presence of a large number of Rho GTPases encoded by the human genome. Here we report the characterization of a new family of Rho GTPases with atypical features. These proteins, which were called Miro-1 and Miro-2 (for mitochondrial Rho), have tandem GTP-binding domains separated by a linker region containing putative calcium-binding EF hand motifs. Genes encoding Miro-like proteins were found in several eukaryotic organisms from Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster to mammals, indicating that these genes evolved early during evolution. Immunolocalization experiments, in which transfected NIH3T3 and COS 7 cells were stained for ectopically expressed Miro as well as for the endogenous Miro-1 protein, showed that Miro was present in mitochondria. Interestingly, overexpression of a constitutively active mutant of Miro-1 (Miro-1/Val-13) induced an aggregation of the mitochondrial network and resulted in an increased apoptotic rate of the cells expressing activated Miro-1. These data indicate a novel role for Rho-like GTPases in mitochondrial homeostasis and apoptosis.
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32.
  • Fransson, Åsa, 1976- (författare)
  • Cell signaling by Rho and Miro GTPases : Studies of Rho GTPases in Cytoskeletal Reorganizations and of Miro GTPases in Mitochondrial Dynamics
  • 2008
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • The Ras superfamily of GTPases embraces six major branches of proteins: the Ras, Rab, Ran, Arf, Rho and Miro subfamilies. The majority of GTPases function as binary switches that cycle between active GTP-bound and inactive GDP-bound states. This thesis will focus primarily on the biological functions of the Rho and Miro proteins. The Rho GTPases control the organization of the actin cytoskeleton and other associated activities, whereas the Miro GTPases are regulators of mitochondrial movement and morphology. A diverse array of cellular phenomena, including cell movement and intracellular membrane trafficking events, are dependent on cytoskeletal rearrangements mediated by Rho GTPases. Although human Rho GTPases are encoded by 20 distinct genes, most studies involving Rho GTPases have focused on the three representatives RhoA, Rac1 and Cdc42, which each regulate specific actin-dependent cellular processes. In an effort to compare the effects of all Rho GTPase members in the same cell system, we transfected constitutively active Rho GTPases in porcine aortic endothelial (PAE) cells and examined their effects on the organization of the actin cytoskeleton. We identified a number of previously undetected roles of the different members of the Rho GTPases. Moreover, we demonstrated that the downstream effectors of Rho GTPases have a broader specificity than previously thought. In a screen for novel Ras-like GTPases, we identified the Miro GTPases (Mitochondrial Rho). In our characterization of Miro, we established that these proteins influence mitochondrial morphology and serve functions in the transport of mitochondria along the microtubule system. Additionally, we provided evidence that Miro can be under control of calcium signaling pathways. Mitochondria are highly dynamic organelles that undergo continuous change in shape and distribution. Defects in mitochondrial dynamics are associated with several neurodegenerative diseases. In conclusion, our findings have contributed to a deeper understanding of the biological roles of Rho and Miro GTPases.
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33.
  • Fransson, Åsa, et al. (författare)
  • The atypical Rho GTPases Miro-1 and Miro-2 have essential roles in mitochondrial trafficking
  • 2006
  • Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 344:2, s. 500-510
  • Tidskriftsartikel (refereegranskat)abstract
    • We recently described the atypical Rho GTPases Miro-1 and Miro-2. These proteins have tandem GTP-binding domains separated by a linker region with putative calcium-binding motives. In addition, the Miro GTPases have a C-terminal transmembrane domain, which confers targeting to the mitochondria. It was reported previously that a constitutively active mutant of Miro-1 induced a clustering of the mitochondria. This response can be separated into two distinct phenotypes: a formation of aggregated mitochondria and the appearance of thread-like mitochondria probably caused by defects in mitochondrial trafficking. The first GTPase domain is required for the clustering of the mitochondria, but the effect is not dependent on the EF-hands. Miro-2 only induces aggregation and not the formation of thread-like mitochondria. Moreover, we show that Miro interacts with the Kinesin-binding proteins, GRIF-1 and OIP106, suggesting that the Miro GTPases form a link between the mitochondria and the trafficking apparatus of the microtubules.
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34.
  • Gad, Annica K. B., et al. (författare)
  • Rho GTPases link cellular contractile force to the density and distribution of nanoscale adhesions
  • 2012
  • Ingår i: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 26:6, s. 2374-2382
  • Tidskriftsartikel (refereegranskat)abstract
    • The ability of cells to adhere and to exert contractile forces governs their capacity to move within an organism. The cytoskeletal regulators of the Rho GTPase proteins are involved in control of the contractile forces of cells. To elucidate the basis of cell migration, we analyzed contractile forces and nanoscale adhesion-related particles in single cells expressing constitutively active variants of Rho GTPases by using traction-force microscopy and ultra-high-resolution stimulated emission depletion microscopy, respectively. RhoAV14 induced large increases in the contractile forces of single cells, with Rac1L61 and RhoDV26 having more moderate effects. The RhoAV14- and RhoDV26-induced forces showed similar spatial distributions and were accompanied by reduced or unaltered cell spreading. In contrast, the Rac1L61-induced force had different, scattered, force distributions that were linked to increased cell spreading. All three of these Rho GTPase activities caused a loss of thick stress fibers and focal adhesions and a more homogenous distribution of nanoscale adhesion-related particles over the ventral surface of the cells. Interestingly, only RhoAV14 increased the density of these particles. Our data suggest a Rac1-specific mode for cells to generate contractile forces. Importantly, increased density and a more homogenous distribution of these small adhesion-related particles promote cellular contractile forces.-Gad, A. K. B., Ronnlund, D., Spaar, A., Savchenko, A. A., Petranyi, G., Blom, H., Szekely, L., Widengren, J., Aspenstrom, P. Rho GTPases link cellular contractile force to the density and distribution of nanoscale adhesions.
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35.
  • Gad, Annica K B, et al. (författare)
  • RhoD regulates cytoskeletal dynamics via the actin nucleation–promoting factor WASp homologue associated with actin Golgi membranes and microtubules
  • 2012
  • Ingår i: Molecular Biology of the Cell. - Stockholm : Karolinska Institutet, Dept of Microbiology, Tumor and Cell Biology. - 1939-4586 .- 1059-1524.
  • Tidskriftsartikel (refereegranskat)abstract
    • The Rho GTPases have mainly been studied in association with their roles in the regulation of actin filament organization. These studies have shown that the Rho GTPases are essential for basic cellular processes, such as cell migration, contraction, and division. In this paper, we report that RhoD has a role in the organization of actin dynamics that is distinct from the roles of the better-studied Rho members Cdc42, RhoA, and Rac1. We found that RhoD binds the actin nucleation–promoting factor WASp homologue associated with actin Golgi membranes and microtubules (WHAMM), as well as the related filamin A–binding protein FILIP1. Of these two RhoD-binding proteins, WHAMM was found to bind to the Arp2/3 complex, while FILIP1 bound filamin A. WHAMM was found to act downstream of RhoD in regulating cytoskeletal dynamics. In addition, cells treated with small interfering RNAs for RhoD and WHAMM showed increased cell attachment and decreased cell migration. These major effects on cytoskeletal dynamics indicate that RhoD and its effectors control vital cytoskeleton-driven cellular processes. In agreement with this notion, our data suggest that RhoD coordinates Arp2/3-dependent and FLNa-dependent mechanisms to control the actin filament system, cell adhesion, and cell migration.
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36.
  • Gizatullina, Zemfira Z., et al. (författare)
  • Effect of transforming growth factor-beta on calcium homeostasis inprostate carcinoma cells.
  • 2003
  • Ingår i: Biochem Biophys Res Commun. ; 304, s. 643-
  • Tidskriftsartikel (refereegranskat)abstract
    • Ca(2+) plays a fundamental role in the control of a variety of cellular functions, in particular, in energy metabolism and apoptosis. In this study, we show that TGF-beta at concentrations of 0.1-1.0 ng/ml transiently increases the level of intracellular Ca(2+) ([Ca(2+)](in)) in human prostate carcinoma, PC-3U, cells. Experiments with mitochondrial inhibitors (oligomycin and antimycin A) and an inhibitor of endoplasmic reticulum Ca(2+) uptake (BHQ) implied that the effect of TGF-beta1 was due to an effect on the mitochondria. TGF-beta1 treatment resulted in a decrease in ATP synthesis and to a depolarisation, leading to a release of Ca(2+) from mitochondria and decreased activity of the Ca(2+) pumps. Analysis of the mitochondria within the PC-3U cells by polarography and membrane potential-sensitive dye (Rhodamine 123) confirmed that under these experimental conditions, TGF-beta1 inhibited ATP synthesis and depolarised the mitochondria. The results implicate that TGF-beta1 affects the function of the mitochondria and may be of significance for the understanding of the proapoptotic effect of TGF-beta1 in these cells.
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37.
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38.
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39.
  • Hertting, Olof, et al. (författare)
  • Vitamin D-deficient mice have more invasive urinary tract infection
  • 2017
  • Ingår i: PLOS ONE. - : Public Library Science. - 1932-6203. ; 12:7
  • Tidskriftsartikel (refereegranskat)abstract
    • Vitamin D deficiency is a common health problem with consequences not limited to bone and calcium hemostasis. Low levels have also been linked to tuberculosis and other respiratory infections as well as autoimmune diseases. We have previously shown that supplementation with vitamin D can induce the antimicrobial peptide cathelicidin during ex vivo infection of human urinary bladder. In rodents, however, cathelicidin expression is not linked to vitamin D and therefore this vitamin D-related effect fighting bacterial invasion is not relevant. To determine if vitamin D had further protective mechanisms during urinary tract infections, we therefore used a mouse model. In vitamin D-deficient mice, we detected more intracellular bacterial communities in the urinary bladder, higher degree of bacterial spread to the upper urinary tract and a skewed cytokine response. Furthermore, we show that the vitamin D receptor was upregulated in the urinary bladder and translocated into the cell nucleus after E. coli infection. This study supports a more general role for vitamin D as a local immune response mediator in the urinary tract.
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40.
  • Holmfeldt, Per, 1973- (författare)
  • Regulation of tubulin heterodimer partitioning during interphase and mitosis
  • 2008
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • The microtubule cytoskeleton, which consists of dynamic polymers of alpha/beta tubulin heterodimers, organizes the cytoplasm and is essential for chromosome segregation during mitosis. My thesis addresses the significance and potential interplay between four distinct microtubule-regulatory proteins. The experimental approach included the development of a replicating vector system directing either constitutive expression of short hairpin RNAs or inducible ectopic expression, which allows stable depletion and/or conditional exchange of gene-products. Based on the originally observed activities in frog egg extracts, MCAK and TOGp have been viewed as major antagonistic proteins that regulate microtubule-dynamics throughout the cell cycle. Surprisingly, while my thesis work confirmed an essential role of these proteins to ensure mitotic fidelity, tubulin subunits partitioning is not controlled by the endogenous levels of MCAK and TOGp in human somatic cells. Our major discovery in these studies is that the activities of both CaMKII and TOGp are essential for spindle bipolarity through a mechanism involving protection of spindle microtubules against MCAK activity at the centrosome. In our search for the major antagonistic activities that regulates microtubule-dynamics in interphase cells, we found that the microtubule-destabilizing activity of Op18 is counteracted by MAP4. These studies also established Op18 and MAP4 as the predominant regulators of tubulin subunit partitioning in all three human cell model systems studied. Moreover, consistent with phosphorylation-inactivation of these two proteins during mitosis, we found that the microtubule-regulatory activities of both MAP4 and Op18 were only evident in interphase cells. Importantly, by employing a system for inducible gene product replacement, we found that site-specific phosphorylation-inactivation of Op18 is the direct cause of the demonstrated hyper-polymerization in response to T-cell antigen receptor triggering. This provides the first formally proven example of a signal transduction pathway for regulation of interphase microtubules. Op18 is frequently upregulated in various types of human malignancies. In addition, a somatic mutation of Op18 has recently been identified in an adenocarcinoma. This thesis work revealed that the mutant Op18 protein exerts increased microtubule-destabilizing activity. The mutant Op18 protein was also shown to be partially resistant to phosphorylation-inactivation during mitosis, which was associated with increased chromosome segregation aberrancies. Interestingly, we also observed the same phenotype by overexpressing the wild type Op18 protein. Thus, either excessive levels of wild type Op18 or normal levels of mutated hyper-active Op18 seems likely to contribute to tumor progression by exacerbating chromosomal instability.
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41.
  • Jaffe, Aron B, et al. (författare)
  • Human CNK1 acts as a scaffold protein, linking Rho and Ras signal transduction pathways
  • 2004
  • Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 24:4, s. 1736-1746
  • Tidskriftsartikel (refereegranskat)abstract
    • Rho family GTPases act as molecular switches to control a variety of cellular responses, including cytoskeletal rearrangements, changes in gene expression, and cell transformation. In the active, GTP-bound state, Rho interacts with an ever-growing number of effector molecules, which promote distinct biochemical pathways. Here, we describe the isolation of hCNK1, the human homologue of Drosophila connector enhancer of ksr, as an effector for Rho. hCNK1 contains several protein-protein interaction domains, and Rho interacts with one of these, the PH domain, in a GTP-dependent manner. A mutant hCNK1, which is unable to bind to Rho, or depletion of endogenous hCNK1 by using RNA interference inhibits Rho-induced gene expression via serum response factor but has no apparent effect on Rho-induced stress fiber formation, suggesting that it acts as a specific effector for transcriptional, but not cytoskeletal, activation pathways. Finally, hCNK1 associates with Rhophilin and RalGDS, Rho and Ras effector molecules, respectively, suggesting that it acts as a scaffold protein to mediate cross talk between the two pathways.
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42.
  • Javadi, Joman, et al. (författare)
  • Syndecan-1 Overexpressing Mesothelioma Cells Inhibit Proliferation, Wound Healing, and Tube Formation of Endothelial Cells
  • 2021
  • Ingår i: Cancers. - : MDPI. - 2072-6694. ; 13:4
  • Tidskriftsartikel (refereegranskat)abstract
    • Simple Summary The transmembrane proteoglycan syndecan-1 (SDC-1) is an important mediator of cell-matrix interactions. The heparan sulfate side-chains of SDC-1 can bind to a multitude of growth factors, cytokines, and chemokines, thereby regulating a plethora of physiological and pathological processes, including angiogenesis. The extracellular region of SDC-1 can be released from the cell surface by the action of sheddases including matrix metalloproteinase-7 and 9, resulting in a soluble protein that is still active and can act as a competitive activator or inhibitor of the cell surface receptor. Accelerated shedding and loss of cell surface SDC-1 is associated with epithelial to mesenchymal transition (EMT) and achievement of a more invasive phenotype in malignant mesothelioma (MM). Transfection with SDC-1 reverts the morphology in epithelioid direction and inhibits the proliferation and migration of MM cells. This study aimed to investigate the role of SDC-1 in angiogenesis. We demonstrate that overexpression and silencing of SDC-1 alters the secretion of angiogenic proteins in MM cells. Upon SDC-1 overexpression, several factors collectively inhibit the proliferation, wound closure, and tube formation of endothelial cells, whereas SDC-1 silencing only affects wound healing. Malignant mesothelioma (MM) is an aggressive tumor of the serosal cavities. Angiogenesis is important for mesothelioma progression, but so far, anti-angiogenic agents have not improved patient survival. Our hypothesis is that better understanding of the regulation of angiogenesis in this tumor would largely improve the success of such a therapy. Syndecan-1 (SDC-1) is a transmembrane heparan sulfate proteoglycan that acts as a co-receptor in various cellular processes including angiogenesis. In MM, the expression of SDC-1 is generally low but when present, SDC-1 associates to epithelioid differentiation, inhibition of tumor cell migration and favorable prognosis, meanwhile SDC-1 decrease deteriorates the prognosis. In the present study, we studied the effect of SDC-1 overexpression and silencing on MM cells ability to secrete angiogenic factors and monitored the downstream effect of SDC-1 modulation on endothelial cells proliferation, wound healing, and tube formation. This was done by adding conditioned medium from SDC-1 transfected and SDC-1 silenced mesothelioma cells to endothelial cells. Moreover, we investigated the interplay and molecular functional changes in angiogenesis in a co-culture system and characterized the soluble angiogenesis-related factors secreted to the conditioned media. We demonstrated that SDC-1 over-expression inhibited the proliferation, wound healing, and tube formation of endothelial cells. This effect was mediated by a multitude of angiogenic factors comprising angiopoietin-1 (Fold change +/- SD: 0.65 +/- 0.07), FGF-4 (1.45 +/- 0.04), HGF (1.33 +/- 0.07), NRG1-beta 1 (1.35 +/- 0.08), TSP-1 (0.8 +/- 0.02), TIMP-1 (0.89 +/- 0.01) and TGF-beta 1 (1.35 +/- 0.01). SDC-1 silencing increased IL8 (1.33 +/- 0.06), promoted wound closure, but did not influence the tube formation of endothelial cells. Pleural effusions from mesothelioma patients showed that Vascular Endothelial Growth Factor (VEGF) levels correlate to soluble SDC-1 levels and have prognostic value. In conclusion, SDC-1 over-expression affects the angiogenic factor secretion of mesothelioma cells and thereby inhibits endothelial cells proliferation, tube formation, and wound healing. VEGF could be used in prognostic evaluation of mesothelioma patients together with SDC-1.
  •  
43.
  • Johansson, Ann-Sofi, 1974- (författare)
  • RhoGTPase Signaling in Cell Polarity and Gene Regulation
  • 2006
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • RhoGTPases are proteins working as molecular switches as they bind and hydrolyze GTP. They are in their active conformation when GTP is bound and are then able to interact with their effector proteins, which relay the downstream signaling. When the GTP is hydrolyzed to GDP, the RhoGTPase is inactivated. RhoGTPases have been shown to be activated by a variety of stimuli and they are implicated in regulation of diverse cellular processes, including cell migration, cell cycle progression, establishment of cell polarity and transformation. We identified mammalian Par6 as a novel effector protein for the RhoGTPases Cdc42 and Rac1. The Caenorhabditis elegans homologue of Par6 had previously been shown to be essential for cell polarity development in the worm embryo. We found that endogenous Par6 colocalized with the tight junction protein ZO-1 in MDCKII epithelial cells. Par6 also interacted with mammalian Par3, another member of the par (for partitioning defective) gene family, first identified in C.elegans. Endogenous Par3 also localized to tight junctions in epithelial cells. This suggested that Par6 and Par3 are part of a complex regulating cell polarity also in mammalian cells. The interaction between Par6 and activated Cdc42 and Rac1 suggested a role for these RhoGTPases in the regulation of this complex.Co-expression of Par6 together with PKCζ, induced a dramatic change in cell morphology. The cells rounded up and long cellular extensions, resembling neurites, were formed. The ability to induce these changes in cell morphology was found to be dependent on the direct interaction between Par6 and PKCζ, as well as on the kinase activity of PKCζ. We observed that cells co-expressing mPar6C and PKCζ contained bundled microtubules and microtubules that hade been acetylated, indicating that the microtubules were stabilized. To investigate the roles of RhoGTPases in PDGF-induced gene expression we performed cDNA microarray analyses on AG01518 human foreskin fibroblasts in which we over-expressed the dominant negative forms of Cdc42, Rac1 and RhoA. We found that the expression of 16 genes, out of the 45 up-regulated by PDGF-BB, were inhibited ≥50% in the presence of dominant negative Cdc42, Rac1 or RhoA. 19 other genes were down-regulated by one or two of the dominant RhoGTPases. Our data implied that the expression of many PDGF-BB induced genes can be affected by RhoGTPase signaling. In conclusion, the work presented here has increased the knowledge of the involvement of RhoGTPase signaling in establishment of cell polarity and gene regulation.
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44.
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45.
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46.
  • Karlsson, Roger, et al. (författare)
  • A chicken beta-actin gene can complement a disruption of the Saccharomyces cerevisiae ACT1 gene
  • 1991
  • Ingår i: Molecular and Cellular Biology. - : American Society for Microbiology. - 0270-7306 .- 1098-5549. ; 11:1, s. 213-217
  • Tidskriftsartikel (refereegranskat)abstract
    • Recently it was demonstrated that beta-actin can be produced in Saccharomyces cerevisiae by using the expression plasmid pY beta actin (R. Karlsson, Gene 68:249-258, 1988), and several site-specific mutants are now being produced in a protein engineering study. To establish a system with which recombinant actin mutants can be tested in vivo and thus enable a correlation to be made with functional effects observed in vitro, a yeast strain lacking endogenous yeast actin and expressing exclusively beta-actin was constructed. This strain is viable but has an altered morphology and a slow-growth phenotype and is temperature sensitive to the point of lethality at 37 degrees C.
  •  
47.
  • Keller, Maureen, et al. (författare)
  • Altered cytoskeletal status in the transition from proneural to mesenchymal glioblastoma subtypes
  • 2022
  • Ingår i: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 12:1, s. 9838-
  • Tidskriftsartikel (refereegranskat)abstract
    • Glioblastoma is a highly aggressive brain tumor with poor patient prognosis. Treatment outcomes remain limited, partly due to intratumoral heterogeneity and the invasive nature of the tumors. Glioblastoma cells invade and spread into the surrounding brain tissue, and even between hemispheres, thus hampering complete surgical resection. This invasive motility can arise through altered properties of the cytoskeleton. We hypothesize that cytoskeletal organization and dynamics can provide important clues to the different malignant states of glioblastoma. In this study, we investigated cytoskeletal organization in glioblastoma cells with different subtype expression profiles, and cytoskeletal dynamics upon subtype transitions. Analysis of the morphological, migratory, and invasive properties of glioblastoma cells identified cytoskeletal components as phenotypic markers that can serve as diagnostic or prognostic tools. We also show that the cytoskeletal function and malignant properties of glioblastoma cells shift during subtype transitions induced by altered expression of the neurodevelopmental transcription factor SOX2. The potential of SOX2 re-expression to reverse the mesenchymal subtype into a more proneural subtype might open up strategies for novel glioblastoma treatments.
  •  
48.
  • Keller, Maureen, et al. (författare)
  • Cytoskeletal Organization Correlates to Motility and Invasiveness of Malignant Mesothelioma Cells
  • 2021
  • Ingår i: Cancers. - : MDPI. - 2072-6694. ; 13:4
  • Tidskriftsartikel (refereegranskat)abstract
    • Malignant mesothelioma (MM) is a rare but highly aggressive cancer that primarily originates from the pleura, peritoneum or pericardium. There is a well-established link between asbestos exposure and progression of MM. Direct invasion of the surrounding tissues is the main feature of MM, which is dependent on dysregulated communication between the mesothelium and the microenvironment. This communication is dependent on the dynamic organization of the cytoskeleton. We have analyzed the organization and function of key cytoskeletal components in MM cell lines of increasing malignancies measured as migratory and invasive properties, and we show that highly malignant and invasive MM cells have an organization of the actin filament and vimentin systems that is distinct from the less malignant MM cell lines. In addition, the Hippo tumor suppressor pathway was inactivated in the invasive MM cells, which was seen as increased YAP nuclear localization.
  •  
49.
  • Lundström, Annika, et al. (författare)
  • Vilse, a conserved Rac/Cdc42 GAP mediating Robo repulsion in tracheal cells and axons
  • 2004
  • Ingår i: Genes & Development. - : Cold Spring Harbor Laboratory. - 0890-9369 .- 1549-5477. ; 18:17, s. 2161-2171
  • Tidskriftsartikel (refereegranskat)abstract
    • Slit proteins steer the migration of many cell types through their binding to Robo receptors, but how Robo controls cell motility is not clear. We describe the functional analysis of vilse, a Drosophila gene required for Robo repulsion in epithelial cells and axons. Vilse defines a conserved family of RhoGAPs (Rho GTPase-activating proteins), with representatives in flies and vertebrates. The phenotypes of vilse mutants resemble the tracheal and axonal phenotypes of Slit and Robo mutants at the CNS midline. Dosage-sensitive genetic interactions between vilse, slit, and robo mutants suggest that vilse is a component of robo signaling. Moreover, overexpression of Vilse in the trachea of robo mutants ameliorates the phenotypes of robo, indicating that Vilse acts downstream of Robo to mediate midline repulsion. Vilse and its human homolog bind directly to the intracellular domains of the corresponding Robo receptors and promote the hydrolysis of RacGTP and, less efficiently, of Cdc42GTP. These results together with genetic interaction experiments with robo, vilse, and rac mutants suggest a mechanism whereby Robo repulsion is mediated by the localized inactivation of Rac through Vilse.
  •  
50.
  • MacAskill, Andrew F., et al. (författare)
  • Miro1 is a calcium sensor for glutamate receptor-dependent localization of mitochondria at synapses.
  • 2009
  • Ingår i: Neuron. - : Elsevier BV. - 0896-6273 .- 1097-4199. ; 61:4, s. 541-555
  • Tidskriftsartikel (refereegranskat)abstract
    • Energy use, mainly to reverse ion movements in neurons, is a fundamental constraint on brain information processing. Trafficking of mitochondria to locations in neurons where there are large ion fluxes is essential for powering neural function. Mitochondrial trafficking is regulated by Ca2+ entry through ionotropic glutamate receptors, but the underlying mechanism is unknown. We show that the protein Miro1 links mitochondria to KIF5 motor proteins, allowing mitochondria to move along microtubules. This linkage is inhibited by micromolar levels of Ca2+ binding to Miro1. With the EF hand domains of Miro1 mutated to prevent Ca2+ binding, Miro1 could still facilitate mitochondrial motility, but mitochondrial stopping induced by glutamate or neuronal activity was blocked. Activating neuronal NMDA receptors with exogenous or synaptically released glutamate led to Miro1 positioning mitochondria at the postsynaptic side of synapses. Thus, Miro1 is a key determinant of how energy supply is matched to energy usage in neurons.
  •  
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