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  • 1. Bagnato, Paola
    et al.
    Castagnino, Alessia
    Cortese, Katia
    Bono, Maria
    Grasso, Silvia
    Bellese, Grazia
    Daniele, Tiziana
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Defilippi, Paola
    Castagnola, Patrizio
    Tacchetti, Carlo
    Cooperative but distinct early co-signaling events originate from ERBB2 and ERBB1 receptors upon trastuzumab treatment in breast cancer cells2017In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 8, no 36, p. 60109-60122Article in journal (Refereed)
    Abstract [en]

    ERBB2 receptor belongs to the ERBB tyrosine kinase receptor family. At variance to the other family members, ERBB2 is a constitutively active orphan receptor. Upon ligand binding and activation, ERBB receptors form homo-or hetero-dimers with the other family members, including ERBB2, promoting an intracellular signaling cascade. ERBB2 is the preferred dimerization partner and ERBB2 heterodimers signaling is stronger and longer acting compared to heterodimers between other ERBB members. The specific contribution of ERBB2 in heterodimer signaling is still undefined. Here we report the formation of circular dorsal ruffles (CDRs) upon treatment of the ERBB2-overexpressing breast cancer cell lines SK-BR-3 and ZR751 with Trastuzumab, a therapeutic humanized monoclonal antibody directed against ERBB2. We found that in SK-BR-3 cells Trastuzumab leads to surface redistribution of ERBB2 and ERBB1 in CDRs, and that the ERBB2-dependent ERK1/2 phosphorylation and ERBB1 expression are both required for CDR formation. In particular, in these cells CDR formation requires activation of both the protein regulator of actin polymerization N-WASP, mediated by ERK1/2, and of the actin depolymerizing protein cofilin, mediated by ERBB1. Furthermore, we suggest that this latter event may be inhibited by the negative cell motility regulator p140Cap, as we found that p140Cap overexpression led to cofilin deactivation and inhibition of CDR formation. In conclusion, here we show for the first time an ERBB2-specific signaling contribution to an ERBB2/ERBB1 heterodimer, in the activation of a complex biological process such as the formation of CDRs.

  • 2. Cortese, Katia
    et al.
    Howes, Mark T.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Tagliatti, Erica
    Bagnato, Paola
    Petrelli, Annalisa
    Bono, Maria
    McMahon, Harvey T.
    Parton, Robert G.
    Tacchetti, Carlo
    The HSP90 inhibitor geldanamycin perturbs endosomal structure and drives recycling ErbB2 and transferrin to modified MVBs/lysosomal compartments2013In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 24, no 2, p. 129-144Article in journal (Refereed)
    Abstract [en]

    The ErbB2 receptor is a clinically validated cancer target whose internalization and trafficking mechanisms remain poorly understood. HSP90 inhibitors, such as geldanamycin (GA), have been developed to target the receptor to degradation or to modulate downstream signaling. Despite intense investigations, the entry route and postendocytic sorting of ErbB2 upon GA stimulation have remained controversial. We report that ErbB2 levels inversely impact cell clathrin-mediated endocytosis (CME) capacity. Indeed, the high levels of the receptor are responsible for its own low internalization rate. GA treatment does not directly modulate ErbB2 CME rate but it affects ErbB2 recycling fate, routing the receptor to modified multivesicular endosomes (MVBs) and lysosomal compartments, by perturbing early/recycling endosome structure and sorting capacity. This activity occurs irrespective of the cargo interaction with HSP90, as both ErbB2 and the constitutively recycled, HSP90-independent, transferrin receptor are found within modified endosomes, and within aberrant, elongated recycling tubules, leading to modified MVBs/lysosomes. We propose that GA, as part of its anticancer activity, perturbs early/recycling endosome sorting, routing recycling cargoes toward mixed endosomal compartments.

  • 3. Daste, Frederic
    et al.
    Walrant, Astrid
    Holst, Mikkel R.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Gadsby, Jonathan R.
    Mason, Julia
    Lee, Ji-Eun
    Brook, Daniel
    Mettlen, Marcel
    Larsson, Elin
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Lee, Steven F.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Gallop, Jennifer L.
    Control of actin polymerization via the coincidence of phosphoinositides and high membrane curvature2017In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 216, no 11, p. 3745-3765Article in journal (Refereed)
    Abstract [en]

    The conditional use of actin during clathrin-mediated endocytosis in mammalian cells suggests that the cell controls whether and how actin is used. Using a combination of biochemical reconstitution and mammalian cell culture, we elucidate a mechanism by which the coincidence of PI(4,5)P-2 and PI(3)P in a curved vesicle triggers actin polymerization. At clathrin-coated pits, PI(3) P is produced by the INPP4A hydrolysis of PI(3,4)P-2, and this is necessary for actin-driven endocytosis. Both Cdc42.guanosine triphosphate and SNX9 activate N-WASP-WIP-and Arp2/3-mediated actin nucleation. Membrane curvature, PI(4,5)P-2, and PI(3) P signals are needed for SNX9 assembly via its PX-BAR domain, whereas signaling through Cdc42 is activated by PI(4,5)P-2 alone. INPP4A activity is stimulated by high membrane curvature and synergizes with SNX9 BAR domain binding in a process we call curvature cascade amplification. We show that the SNX9-driven actin comets that arise on human disease-associated oculocerebrorenal syndrome of Lowe (OCRL) deficiencies are reduced by inhibiting PI(3) P production, suggesting PI(3) P kinase inhibitors as a therapeutic strategy in Lowe syndrome.

  • 4. Daumke, Oliver
    et al.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Vallis, Yvonne
    Martens, Sascha
    Butler, P Jonathan G
    McMahon, Harvey T
    Architectural and mechanistic insights into an EHD ATPase involved in membrane remodelling2007In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 449, no 7164, p. 923-927Article in journal (Refereed)
    Abstract [en]

    The ability to actively remodel membranes in response to nucleotide hydrolysis has largely been attributed to GTPases of the dynamin superfamily, and these have been extensively studied. Eps15 homology (EH)-domain-containing proteins (EHDs/RME-1/pincher) comprise a less-well-characterized class of highly conserved eukaryotic ATPases implicated in clathrin-independent endocytosis, and recycling from endosomes. Here we show that EHDs share many common features with the dynamin superfamily, such as a low affinity for nucleotides, the ability to tubulate liposomes in vitro, oligomerization around lipid tubules in ring-like structures and stimulated nucleotide hydrolysis in response to lipid binding. We present the structure of EHD2, bound to a non-hydrolysable ATP analogue, and provide evidence consistent with a role for EHDs in nucleotide-dependent membrane remodelling in vivo. The nucleotide-binding domain is involved in dimerization, which creates a highly curved membrane-binding region in the dimer. Oligomerization of dimers occurs on another interface of the nucleotide-binding domain, and this allows us to model the EHD oligomer. We discuss the functional implications of the EHD2 structure for understanding membrane deformation.

  • 5. Doherty, Gary J
    et al.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    GRAF1-dependent endocytosis2009In: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 37, no 5, p. 1061-1065Article in journal (Refereed)
    Abstract [en]

    The role of endocytosis in controlling a multitude of cell biological events is well established. Molecular and mechanistic characterization of endocytosis has predominantly focused on CME (clathrin-mediated endocytosis), although many other endocytic pathways have been described. it was recently shown that the BAR (Bin/amphiphysin/Rvs) and Rho GAP (GTPase-activating protein) domain-containing protein GRAF1 (GTPase regulator associated with focal adhesion kinase-1) is found on prevalent, pleiomorphic endocytic membranes, and is essential for the major, clathrin-independent endocytic pathway that these membranes mediate. This pathway is characterized by its ability to internalize GPI (glycosylphosphatidylinositol)anchored proteins, bacterial toxins and large amounts of extracellular fluid. These membrane carriers are highly dynamic and associated with the activity of the small G-protein Cdc42 (cell division cycle 42). in the present paper, we review the role of GRAF1 in this CLIC (clathrin-independent carrier)/GEEC (GPI-anchored protein-enriched early endocytic compartment) endocytic pathway and discuss the current understanding regarding how this multidomain protein functions at the interface between membrane sculpting, small G-protein signalling and endocytosis.

  • 6. Doherty, Gary J.
    et al.
    Åhlund, Monika K.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Howes, Mark T.
    Moren, Bjorn
    Parton, Robert G.
    McMahon, Harvey T.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The endocytic protein GRAF1 is directed to cell-matrix adhesion sites and regulates cell spreading2011In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 22, no 22, p. 4380-4389Article in journal (Refereed)
    Abstract [en]

    The rho GTPase-activating protein GTPase regulator associated with focal adhesion kinase-1 (GRAF1) remodels membranes into tubulovesicular clathrin-independent carriers (CLICs) mediating lipid-anchored receptor endocytosis. However, the cell biological functions of this highly prevalent endocytic pathway are unclear. In this article, we present biochemical and cell biological evidence that GRAF1 interacted with a network of endocytic and adhesion proteins and was found enriched at podosome-like adhesions and src-induced podosomes. We further demonstrate that these sites comprise microdomains of highly ordered lipid enriched in GRAF1 endocytic cargo. GRAF1 activity was upregulated in spreading cells and uptake via CLICs was concentrated at the leading edge of migrating cells. Depletion of GRAF1, which inhibits CLIC generation, resulted in profound defects in cell spreading and migration. We propose that GRAF1 remodels membrane microdomains at adhesion sites into endocytic carriers, facilitating membrane turnover during cell morphological changes.

  • 7. Eberth, Alexander
    et al.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gremer, Lothar
    Dvorsky, Radovan
    Koessmeier, Katja T
    McMahon, Harvey T
    Ahmadian, Mohammad Reza
    A BAR domain-mediated autoinhibitory mechanism for RhoGAPs of the GRAF family2009In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 417, no 1, p. 371-377Article in journal (Refereed)
    Abstract [en]

    The BAR (Bin/amphiphysin/Rvs) domain defines an emerging superfamily of proteins implicated in fundamental biological processes by sensing and inducing membrane curvature. We identified a novel autoregulatory function for the BAR domain of two related GAPs' (GTPase-activating proteins) of the GRAF (GTPase regulator associated with focal adhesion kinase) subfamily. We demonstrate that the N-terminal fragment of these GAPs including the BAR domain interacts directly with the GAP domain and inhibits its activity. Analysis of various BAR and GAP domains revealed that the BAR domain-mediated inhibition of these GAPs' function is highly specific. These GAPs, in their autoinhibited state, are able to bind and tubulate liposomes in vitro, and to generate lipid tubules in cells. Taken together, we identified BAR domains as cis-acting inhibitory elements that very likely mask the active sites of the GAP domains and thus prevent down-regulation of Rho proteins. Most remarkably, these BAR proteins represent a dual-site system with separate membrane-tubulation and GAP-inhibitory functions that operate simultaneously.

  • 8.
    Francis, Monika K.
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Holst, Mikkel R.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Vidal-Quadras, Maite
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Henriksson, Sara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Santarella-Mellwig, Rachel
    Sandblad, Linda
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Endocytic membrane turnover at the leading edge is driven by a transient interaction between Cdc42 and GRAF12015In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 128, no 22, p. 4183-4195Article in journal (Refereed)
    Abstract [en]

    Changes in cell morphology require coordination of plasma membrane turnover and cytoskeleton dynamics, processes that are regulated by Rho GTPases. Here, we describe how a direct interaction between the Rho GTPase Cdc42 and the GTPase activating protein (GAP) GRAF1, facilitate rapid cell surface turnover at the leading edge. Both Cdc42 and GRAF1 were required for fluid phase uptake and regulated the generation of transient GRAF1-coated endocytic carriers, distinct from clathrin coated vesicles. GRAF1 was found to transiently assemble at discrete Cdc42-enriched punctae at the plasma membrane resulting in a corresponding decrease in Cdc42 microdomain association. However, Cdc42 captured in its active state was, via a GAP domain mediated interaction, localised together with GRAF1 on accumulated internal structures derived from the cell surface. Correlative fluorescence and electron tomography microscopy revealed that these structures were clusters of small membrane carriers affected in their endosomal processing. We conclude that a transient interaction between Cdc42 and GRAF1 drives endocytic turnover and controls the transition essential for endosomal maturation of plasma membrane internalised by this mechanism.

  • 9.
    Francis, Monika K.
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Krupp, Nikolai
    Blomberg, Jeanette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Behrmann, Elmar
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    GRAF1 sculpts membrane through a regulated oligomerisation reactionManuscript (preprint) (Other academic)
  • 10. Hoernke, M.
    et al.
    Mohan, J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Larsson, Elin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Kahra, Dana
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Westenhoff, S.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Schwieger, C.
    Determining membrane bound protein structures by infrared reflection-absorption spectroscopy2017In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 46, p. S161-S161Article in journal (Other academic)
  • 11. Hoernke, M
    et al.
    Mohan, Jagan
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Larsson, Elin
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Blomberg, J
    Kahra, Dana
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Daumke, O
    Westenhof, S
    Schweiger, C
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    TP driven stabilization of a membrane bound open confirmation of the ATPase EHD2 restrains caveolae dynamicsManuscript (preprint) (Other academic)
  • 12. Hoernke, Maria
    et al.
    Larsson, Elin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mohan, Jagan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Blomberg, Jeanette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Westenhoff, Sebastian
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schwieger, Christian
    Structural Mechanism in a Membrane Remodelling ATP-ASE2016In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, no 3, p. 578A-578AArticle in journal (Other academic)
  • 13. Hoernke, Maria
    et al.
    Mohan, Jagan
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Larsson, Elin
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Blomberg, Jeanette
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Kahra, Dana
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Westenhoff, Sebastian
    Schwieger, Christian
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    EHD2 restrains dynamics of caveolae by an ATP-dependent, membrane-bound, open conformation2017In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 22, p. E4360-E4369Article in journal (Refereed)
    Abstract [en]

    The EH-domain-containing protein 2 (EHD2) is a dynamin-related ATPase that confines caveolae to the cell surface by restricting the scission and subsequent endocytosis of these membrane pits. For this, EHD2 is thought to first bind to the membrane, then to oligomerize, and finally to detach, in a stringently regulated mechanistic cycle. It is still unclear how ATP is used in this process and whether membrane binding is coupled to conformational changes in the protein. Here, we show that the regulatory N-terminal residues and the EH domain keep the EHD2 dimer in an autoinhibited conformation in solution. By significantly advancing the use of infrared reflection-absorption spectroscopy, we demonstrate that EHD2 adopts an open conformation by tilting the helical domains upon membrane binding. We show that ATP binding enables partial insertion of EHD2 into the membrane, where G-domain-mediated oligomerization occurs. ATP hydrolysis is related to detachment of EHD2 from the membrane. Finally, we demonstrate that the regulation of EHD2 oligomerization in a membrane-bound state is crucial to restrict caveolae dynamics in cells.

  • 14.
    Holst, Mikkel Roland
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Vidal-Quadras, Maite
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Larsson, Elin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Song, Jie
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Hubert, Madlen
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Blomberg, Jeanette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Lundborg, Magnus
    Landström, Maréne
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Clathrin-Independent Endocytosis Suppresses Cancer Cell Blebbing and Invasion2017In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 20, no 8, p. 1893-1905Article in journal (Refereed)
    Abstract [en]

    Cellular blebbing, caused by local alterations in cellsurface tension, has been shown to increase the invasiveness of cancer cells. However, the regulatory mechanisms balancing cell-surface dynamics and bleb formation remain elusive. Here, we show that an acute reduction in cell volume activates clathrinindependent endocytosis. Hence, a decrease in surface tension is buffered by the internalization of the plasma membrane (PM) lipid bilayer. Membrane invagination and endocytosis are driven by the tension- mediated recruitment of the membrane sculpting and GTPase-activating protein GRAF1 (GTPase regulator associated with focal adhesion kinase-1) to the PM. Disruption of this regulation by depleting cells of GRAF1 or mutating key phosphatidylinositol- interacting amino acids in the protein results in increased cellular blebbing and promotes the 3D motility of cancer cells. Our data support a role for clathrin-independent endocytic machinery in balancing membrane tension, which clarifies the previously reported role of GRAF1 as a tumor suppressor.

  • 15. Howes, Mark T
    et al.
    Kirkham, Matthew
    Riches, James
    Cortese, Katia
    Walser, Piers J
    Simpson, Fiona
    Hill, Michelle M
    Jones, Alun
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. MRC Laboratory of Molecular Biology, Cambridge, England.
    Lindsay, Margaret R
    Hernandez-Deviez, Delia J
    Hadzic, Gordana
    McCluskey, Adam
    Bashir, Rumasia
    Liu, Libin
    Pilch, Paul
    McMahon, Harvey
    Robinson, Phillip J
    Hancock, John F
    Mayor, Satyajit
    Parton, Robert G
    Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells2010In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 190, no 4, p. 675-691Article in journal (Refereed)
    Abstract [en]

    Although the importance of clathrin- and caveolin-independent endocytic pathways has recently emerged, key aspects of these routes remain unknown. Using quantitative ultrastructural approaches, we show that clathrin-independent carriers (CLICs) account for approximately three times the volume internalized by the clathrin-mediated endocytic pathway, forming the major pathway involved in uptake of fluid and bulk membrane in fibroblasts. Electron tomographic analysis of the 3D morphology of the earliest carriers shows that they are multidomain organelles that form a complex sorting station as they mature. Proteomic analysis provides direct links between CLICs, cellular adhesion turnover, and migration. Consistent with this, CLIC-mediated endocytosis of key cargo proteins, CD44 and Thy-1, is polarized at the leading edge of migrating fibroblasts, while transient ablation of CLICs impairs their ability to migrate. These studies provide the first quantitative ultrastructural analysis and molecular characterization of the major endocytic pathway in fibroblasts, a pathway that provides rapid membrane turnover at the leading edge of migrating cells.

  • 16.
    Hubert, Madlen
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Larsson, Elin
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Caveolae dynamics is strongly influenced by the lipid composition of the plasma membrane2017In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 46, p. S121-S121Article in journal (Other academic)
  • 17.
    Håberg, Karin
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Carlsson, Sven
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    SNX18 is an SNX9 paralog that acts as a membrane tubulator in AP-1-positive endosomal trafficking.2008In: Journal of Cell Science, ISSN 0021-9533, Vol. 121, no Pt 9, p. 1495-505Article in journal (Refereed)
  • 18.
    Kindstedt, Elin
    et al.
    Umeå University, Faculty of Medicine, Department of Odontology.
    Koskinen Holm, Cecilia
    Umeå University, Faculty of Medicine, Department of Odontology.
    Sulniute, Rima
    Umeå University, Faculty of Medicine, Department of Odontology.
    Martinez-Carrasco, Irene
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Lundberg, Pernilla
    Umeå University, Faculty of Medicine, Department of Odontology.
    CCL11, a novel mediator of inflammatory bone resorption2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, no 1, article id 5334Article in journal (Refereed)
    Abstract [en]

    Normal bone homeostasis, which is regulated by bone-resorbing osteoclasts and bone-forming osteoblasts is perturbed by inflammation. Inchronic inflammatory disease with disturbed bone remodelling, e.g. rheumatoid arthritis, patients show increased serum levels of the chemokine eotaxin-1 (CCL11). Herein, we demonstrate an inflammatory driven expression of CCL11 in bone tissue and a novel role of CCL11 in osteoclast migration and resorption. Using an inflammatory bone lesion model and primary cell cultures, we discovered that osteoblasts express CCL11 in vivo and in vitro and that expression increased during inflammatory conditions. Osteoclasts did not express CCL11, but the high affinity receptor CCR3 was significantly upregulated during osteoclast differentiation and found to colocalise with CCL11. Exogenous CCL11 was internalised in osteoclast and stimulated the migration of pre-osteoclast and concomitant increase in bone resorption. Our data pinpoints that the CCL11/CCR3 pathway could be a new target for treatment of inflammatory bone resorption.

  • 19.
    Kong, Ziqing
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Jia, Shaodong
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Anna Lena
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Appelblad, Patrik
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience. Merck Chemicals and Life Science AB, Solna, Sweden.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Moritz, Thomas
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Simultaneous determination of ribonucleoside and deoxyribonucleoside triphosphates in biological samples by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry2018In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 11, article id e66Article in journal (Refereed)
    Abstract [en]

    Information about the intracellular concentration of dNTPs and NTPs is important for studies of the mechanisms of DNA replication and repair, but the low concentration of dNTPs and their chemical similarity to NTPs present a challenge for their measurement. Here, we describe a new rapid and sensitive method utilizing hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry for the simultaneous determination of dNTPs and NTPs in biological samples. The developed method showed linearity (R2 > 0.99) in wide concentration ranges and could accurately quantify dNTPs and NTPs at low pmol levels. The intra-day and inter-day precision were below 13%, and the relative recovery was between 92% and 108%. In comparison with other chromatographic methods, the current method has shorter analysis times and simpler sample pre-treatment steps, and it utilizes an ion-pair-free mobile phase that enhances mass-spectrometric detection. Using this method, we determined dNTP and NTP concentrations in actively dividing and quiescent mouse fibroblasts.

  • 20.
    Larsson, Elin
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Morén, Björn
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Shah, Claudio
    Max-Delbrück-Center for Molecular Medicine.
    Daumke, Oliver
    Max-Delbrück-Center for Molecular Medicine.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    An EH-domain switching mechanism regulates stable membrane association of EHD2Manuscript (preprint) (Other academic)
    Abstract [en]

    EHD2 is a dimeric ATPase known to stabilise the surface connection of the characteristic small invaginations of the cell surface termed caveolae. EHD2 oligomerises into rings around lipid membranes thereby controlling their shape. Here, we have analysed the domain interactions and mechanism that control the stable membrane association of EHD2 at caveolae. We have found that the N-terminus of EHD2, which is buried in the core protein and obstruct assembly, has to be relieved by an EH domain dependent mechanism. The binding between the EH domain and a loop in the GTPase domain of EHD2 was required for stable membrane association, but the loop in itself was not sufficient for specific recruitment to caveolae. A positively charged stretch in the EH domain is proposed to bind to lipids and thereby influence the exchange rate of EHD2. Taken together, we propose a stringent regulatory mechanism for the assembly of EHD2 involving switching of the EH domain position to release the N-terminus and facilitate oligomerisation.

  • 21.
    Lundmark, Richard
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Carlsson, Sven
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sorting nexin 9 participates in clathrin-mediated endocytosis through interactions with the core components2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 47, p. 46772-46781Article in journal (Refereed)
    Abstract [en]

    Sorting nexin 9 (SNX9) belongs to a family of proteins, the sorting nexins, that are characterized by the presence of a subclass of the phosphoinositide-binding phox domain. SNX9 has in its amino terminus a Src homology 3 domain and a region with predicted low complexity followed by a carboxyl-terminal part containing the phox domain. We previously found that SNX9 is one of the major proteins in hematopoietic cells that binds to the alpha and beta2-appendages of adaptor protein complex 2 (AP-2), a protein with a critical role in the formation of clathrin-coated vesicles at the plasma membrane. In the present study we show that clathrin and dynamin-2, two other essential molecules in the endocytic process, also interact with SNX9. We found that both AP-2 and clathrin bind to the low complexity region in SNX9 in a cooperative manner, whereas dynamin-2 binds to the Src homology 3 domain. In the cytosol, SNX9 is present in a 14.5 S complex containing dynamin-2 and an unidentified 41-kDa protein. In HeLa cells, SNX9 co-localized with both AP-2 and dynamin-2 at the plasma membrane or on vesicular structures derived from it but not with the early endosomal marker EEA1 or with AP-1. The results suggest that SNX9 may be recruited together with dynamin-2 and become co-assembled with AP-2 and clathrin at the plasma membrane. Overexpression in both K562 and HeLa cells of truncated forms of SNX9 interfered with the uptake of transferrin, consistent with a role of SNX9 in endocytosis.

  • 22.
    Lundmark, Richard
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Carlsson, Sven
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The beta-appendages of the four adaptor-protein (AP) complexes: structure and binding properties, and identification of sorting nexin 9 as an accessory protein to AP-22002In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 362, no 3, p. 597-607Article in journal (Refereed)
    Abstract [en]

    Adaptor protein (AP) complexes are essential components for the formation of coated vesicles and the recognition of cargo proteins for intracellular transport. Each AP complex exposes two appendage domains with that function to bind regulatory accessory proteins in the cytosol. Secondary structure predictions, sequence alignments and CD spectroscopy were used to relate the beta-appendages of all human AP complexes to the previously published crystal structure of AP-2. The results suggested that the beta-appendages of AP-1, AP-2 and AP-3 have similar structures, consisting of two subdomains, whereas that of AP-4 lacks the inner subdomain. Pull-down and overlay assays showed partial overlap in the binding specificities of the beta-appendages of AP-1 and AP-2, whereas the corresponding domain of AP-3 displayed a unique binding pattern. That AP-4 may have a truncated, non-functional domain was indicated by its apparent inability to bind any proteins from cytosol. Of several novel beta-appendage-binding proteins detected, one that had affinity exclusively for AP-2 was identified as sorting nexin 9 (SNX9). SNX9, which contains a phox and an Src homology 3 domain, was found in large complexes and was at least partially associated with AP-2 in the cytosol. SNX9 may function to assist AP-2 in its role at the plasma membrane.

  • 23.
    Lundmark, Richard
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Carlsson, Sven R
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Driving membrane curvature in clathrin-dependent and clathrin-independent endocytosis.2010In: Seminars in Cell and Developmental Biology, ISSN 1084-9521, E-ISSN 1096-3634, Vol. 21, no 4, p. 363-70Article in journal (Refereed)
    Abstract [en]

    Cellular activity depends to a large extent on membrane bilayer dynamics. Many processes, such as organelle biogenesis and vesicular transport, rely on alterations in membrane structure and shape. It is now widely accepted that intracellular membrane curvature generation and remodelling is mediated and regulated by protein action, and the mechanisms behind the processes are currently being revealed. Here, we will briefly discuss the key principles of membrane deformation and focus on different endocytic events that use various kinds of proteins to shape the plasma membrane into transport carriers. The entry routes are adopted to make sure that a vast variety of molecules on the cell surface can be regulated by endocytosis. The principles for membrane sculpting of endocytic carriers can be viewed either from a perspective of rigid coat budding or of flexible opportunistic budding. We will discuss these principles and their implications, focusing on clathrin-dependent and -independent carrier formation and the proteins involved in the respective pathways.

  • 24.
    Lundmark, Richard
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Carlsson, Sven R
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Expression and properties of sorting nexin 9 in dynamin-mediated endocytosis2005In: Methods in Enzymology, ISSN 0076-6879, E-ISSN 1557-7988, Vol. 404, p. 545-556Article in journal (Refereed)
    Abstract [en]

    Sorting nexin 9 (SNX9) is identified as an important regulator of dynamin function in clathrin-mediated endocytosis. SNX9 recruits dynamin to the plasma membrane and promotes its GTPase activity, resulting in membrane constriction and ultimate transport vesicle scission. This chapter describes procedures to express recombinant SNX9, to biochemically characterize the cytosolic complex between SNX9 and dynamin, and to identify additional interacting partners of SNX9. Assays are presented to investigate the requirements for SNX9-dependent membrane recruitment of dynamin in vitro and in vivo.

  • 25.
    Lundmark, Richard
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Carlsson, Sven R
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Regulated membrane recruitment of dynamin-2 mediated by sorting nexin 9.2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 41, p. 42694-42702Article in journal (Refereed)
    Abstract [en]

    The endocytic proteins sorting nexin 9 (SNX9) and dynamin-2 (Dyn2) assemble in the cytosol as a resting complex, together with a 41-kDa protein. We show here that the complex can be activated for membrane binding of SNX9 and Dyn2 by incubation of cytosol in the presence of ATP. SNX9 was essential for Dyn2 recruitment, whereas the reverse was not the case. RNA interference experiments confirmed that SNX9 functions as a mediator of Dyn2 recruitment to membranes in cells. The 41-kDa component was identified as the glycolytic enzyme aldolase. Aldolase bound with high affinity to a tryptophan-containing acidic sequence in SNX9 located close to its Phox homology domain, thereby blocking the membrane binding activity of SNX9. Phosphorylation of SNX9 released aldolase from the native cytosolic complex and rendered SNX9 competent for membrane binding. The results suggest that SNX9-dependent recruitment of Dyn2 to the membrane is regulated by an interaction between SNX9 and aldolase.

  • 26.
    Lundmark, Richard
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Carlsson, Sven R
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    SNX9 - a prelude to vesicle release2009In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 122, no 1, p. 5-11Article in journal (Refereed)
    Abstract [en]

    The sorting nexin SNX9 has, in the past few years, been singled out as an important protein that participates in fundamental cellular activities. SNX9 binds strongly to dynamin and is partly responsible for the recruitment of this GTPase to sites of endocytosis. SNX9 also has a high capacity for modulation of the membrane and might therefore participate in the formation of the narrow neck of endocytic vesicles before scission occurs. Once assembled on the membrane, SNX9 stimulates the GTPase activity of dynamin to facilitate the scission reaction. It has also become clear that SNX9 has the ability to activate the actin regulator N-WASP in a membrane-dependent manner to coordinate actin polymerization with vesicle release. In this Commentary, we summarize several aspects of SNX9 structure and function in the context of membrane remodeling, discuss its interplay with various interaction partners and present a model of how SNX9 might work in endocytosis.

  • 27.
    Lundmark, Richard
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Doherty, Gary J
    Howes, Mark T
    Cortese, Katia
    Vallis, Yvonne
    Parton, Robert G
    McMahon, Harvey T
    The GTPase-activating protein GRAF1 regulates the CLIC/GEEC endocytic pathway2008In: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 18, no 22, p. 1802-1808Article in journal (Refereed)
    Abstract [en]

    Clathrin-independent endocytosis is an umbrella term for a variety of endocytic pathways that internalize numerous cargoes independently of the canonical coat protein Clathrin [1, 2]. Electron-microscopy studies have defined the pleiomorphic CLathrin-Independent Carriers (CLICs) and GPI-Enriched Endocytic Compartments (GEECs) as related major players in such uptake [3, 4]. This CLIC/GEEC pathway relies upon cellular signaling and activation through small G proteins, but mechanistic insight into the biogenesis of its tubular and tubulovesicular carriers is lacking. Here we show that the Rho-GAP-domain-containing protein GRAF1 marks, and is indispensable for, a major Clathrin-independent endocytic pathway. This pathway is characterized by its ability to internalize bacterial exotoxins, GPI-linked proteins, and extracellular fluid. We show that GRAF1 localizes to PtdIns(4,5)P2-enriched, tubular, and punctate lipid structures via N-terminal BAR and PH domains. These membrane carriers are relatively devoid of caveolin1 and flotillin1 but are associated with activity of the small G protein Cdc42. This study provides the first specific noncargo marker for CLIC/GEEC endocytic membranes and demonstrates how GRAF1 can coordinate small G protein signaling and membrane remodeling to facilitate internalization of CLIC/GEEC pathway cargoes.

  • 28.
    Lundmark, Richard
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Doherty, Gary J
    Vallis, Yvonne
    Peter, Brian J
    McMahon, Harvey T
    Arf family GTP loading is activated by, and generates, positive membrane curvature.2008In: The Biochemical journal, ISSN 1470-8728, Vol. 414, no 2, p. 189-94Article in journal (Refereed)
    Abstract [en]

    Small G-proteins belonging to the Arf (ADP-ribosylation factor) family serve as regulatory proteins for numerous cellular processes through GTP-dependent recruitment of effector molecules. In the present study we demonstrate that proteins in this family regulate, and are regulated by, membrane curvature. Arf1 and Arf6 were shown to load GTP in a membrane-curvature-dependent manner and stabilize, or further facilitate, changes in membrane curvature through the insertion of an amphipathic helix.

  • 29. Marchès, O
    et al.
    Batchelor, M
    Shaw, RK
    Patel, A
    Cummings, N
    Nagai, T
    Sasakawa, C
    Carlsson, Sven R
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Cougoule, C
    Caron, E
    Knutton, S
    Connerton, I
    Frankel, G
    EspF of enteropathogenic Escherichia coli binds sorting nexin 92006In: Journal of Bacteriology, Vol. 188, no 8, p. 3110-3115Article in journal (Refereed)
  • 30. Melo, Arthur Alves
    et al.
    Hegde, Balachandra G.
    Shah, Claudio
    Larsson, Elin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Isas, J. Mario
    Kunz, Severine
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Langen, Ralf
    Daumke, Oliver
    Structural insights into the activation mechanism of dynamin-like EHD ATPases2017In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 22, p. 5629-5634Article in journal (Refereed)
    Abstract [en]

    Eps15 (epidermal growth factor receptor pathway substrate 15)homology domain containing proteins (EHDs) comprise a family of dynamin-related mechano-chemical ATPases involved in cellular membrane trafficking. Previous studies have revealed the structure of the EHD2 dimer, but the molecular mechanisms of membrane recruitment and assembly have remained obscure. Here, we determined the crystal structure of an amino-terminally truncated EHD4 dimer. Compared with the EHD2 structure, the helical domains are 50 degrees rotated relative to the GTPase domain. Using electron paramagnetic spin resonance (EPR), we show that this rotation aligns the two membrane-binding regions in the helical domain toward the lipid bilayer, allowing membrane interaction. A loop rearrangement in GTPase domain creates a new interface for oligomer formation. Our results suggest that the EHD4 structure represents the active EHD conformation, whereas the EHD2 structure is autoinhibited, and reveal a complex series of domain rearrangements accompanying activation. A comparison with other peripheral membrane proteins elucidates common and specific features of this activation mechanism.

  • 31.
    Mohan, Jagan
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Hubert, Madlen
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Larsson, Elin
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Schweiger, C
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Structural arrangement of membrane-bound cavinManuscript (preprint) (Other academic)
  • 32.
    Mohan, Jagan
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Morén, Björn
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Larsson, Elin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Holst, Mikkel
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Cavin3 interacts with cavin1 and caveolin1 to increase surface dynamics of caveolae2015In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 128, no 5, p. 979-991Article in journal (Refereed)
    Abstract [en]

    Caveolae are invaginations of the cell surface thought to regulate membrane tension, signalling, adhesion and lipid homeostasis due to their dynamic behaviour ranging from stable surface association to dynamic rounds of fission and fusion with the plasma membrane. The caveolae coat is generated by oligomerisation of the membrane protein caveolin and the family of cavin proteins. Here, we show that cavin3 is targeted to caveolae by cavin1 where it interacts with the scaffolding domain of caveolin1 and promote caveolae dynamics. We found that the N-terminal region of cavin3 binds a trimer of the cavin1 N-terminus in competition with a homologous cavin2 region, showing that the cavins form distinct subcomplexes via their N-terminal regions. Our data shows that cavin3 is enriched at deeply invaginated caveolae and that loss of cavin3 in cells results in an increase of stable caveolae and a decrease of caveolae with short duration time at the membrane. We propose that cavin3 is recruited to the caveolae coat by cavin1 to interact with caveolin1 and regulate the duration time of caveolae at the plasma membrane.

  • 33.
    Moodie, Lindon W. K.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hubert, Madlen
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Zhou, Xin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Albers, Michael Franz
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hedberg, Christian
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Photoactivated Colibactin Probes Induce Cellular DNA Damage2019In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 58, no 5, p. 1417-1421Article in journal (Refereed)
    Abstract [en]

    Colibactin is a small molecule produced by certain bacterial species of the human microbiota that harbour the pks genomic island. Pks(+) bacteria induce a genotoxic phenotype in eukaryotic cells and have been linked with colorectal cancer progression. Colibactin is produced in a benign, prodrug form which, prior to export, is enzymatically matured by the producing bacteria to its active form. Although the complete structure of colibactin has not been determined, key structural features have been described including an electrophilic cyclopropane motif, which is believed to alkylate DNA. To investigate the influence of the putative "warhead" and the prodrug strategy on genotoxicity, a series of photolabile colibactin probes were prepared that upon irradiation induced a pks(+) like phenotype in HeLa cells. Furthermore, results from DNA cross-linking and imaging studies of clickable analogues enforce the hypothesis that colibactin effects its genotoxicity by directly targeting DNA.

  • 34. Moren, Bjorn
    et al.
    Hansson, Bjorn
    Negoita, Florentina
    Fryklund, Claes
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Goransson, Olga
    Stenkula, Karin G.
    EHD2 regulates adipocyte function and is enriched at cell surface-associated lipid droplets in primary human adipocytes2019In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 30, no 10, p. 1147-1159Article in journal (Refereed)
    Abstract [en]

    Adipocytes play a central role in energy balance, and dysfunctional adipose tissue severely affects systemic energy homeostasis. The ATPase EH domain-containing 2 (EHD2) has previously been shown to regulate caveolae, plasma membrane-specific domains that are involved in lipid uptake and signal transduction. Here, we investigated the role of EHD2 in adipocyte function. We demonstrate that EHD2 protein expression is highly up-regulated at the onset of triglyceride accumulation during adipocyte differentiation. Small interfering RNA-mediated EHD2 silencing affected the differentiation process and impaired insulin sensitivity, lipid storage capacity, and lipolysis. Fluorescence imaging revealed localization of EHD2 to caveolae, close to cell surface-associated lipid droplets in primary human adipocytes. These lipid droplets stained positive for glycerol transporter aquaporin 7 and phosphorylated perilipin-1 following adrenergic stimulation. Further, EHD2 overexpression in human adipocytes increased the lipolytic signaling and suppressed the activity of transcription factor PPAR.. Overall, these data suggest that EHD2 plays a key role for adipocyte function.

  • 35.
    Morén, Björn
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Shah, Claudio
    Howes, Mark T
    Schieber, Nicole L
    McMahon, Harvey T
    Parton, Robert G
    Daumke, Oliver
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    EHD2 regulates caveolar dynamics via ATP-driven targeting and oligomerization2012In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 23, no 7, p. 1316-1329Article in journal (Refereed)
    Abstract [en]

    Eps15 homology domain-containing 2 (EHD2) belongs to the EHD-containing protein family of dynamin-related ATPases involved in membrane remodeling in the endosomal system. EHD2 dimers oligomerize into rings on highly curved membranes, resulting in stimulation of the intrinsic ATPase activity. In this paper, we report that EHD2 is specifically and stably associated with caveolae at the plasma membrane and not involved in clathrin-mediated endocytosis or endosomal recycling, as previously suggested. EHD2 interacts with pacsin2 and cavin1, and ordered membrane assembly of EHD2 is dependent on cavin1 and caveolar integrity. While the EHD of EHD2 is dispensable for targeting, we identified a loop in the nucleotide-binding domain that, together with ATP binding, is required for caveolar localization. EHD2 was not essential for the formation or shaping of caveolae, but high levels of EHD2 caused distortion and loss of endogenous caveolae. Assembly of EHD2 stabilized and constrained caveolae to the plasma membrane to control turnover, and depletion of EHD2, resulting in endocytic and more dynamic and short-lived caveolae. Thus, following the identification of caveolin and cavins, EHD2 constitutes a third structural component of caveolae involved in controlling the stability and turnover of this organelle.

  • 36.
    Nygård Skalman, Lars
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Holst, Mikkel R.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Larsson, Elin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Plasma membrane damage caused by listeriolysin O is not repaired through endocytosis of the membrane pore2018In: Biology Open, ISSN 2046-6390, Vol. 7, no 10Article in journal (Refereed)
    Abstract [en]

    Endocytic mechanisms have been suggested to be important for plasma membrane repair in response to pore-forming toxins such as listeriolysin O (LLO), which form membrane pores that disrupt cellular homeostasis. Yet, little is known about the specific role of distinct endocytic machineries in this process. Here, we have addressed the importance of key endocytic pathways and developed reporter systems for real-time imaging of the endocytic response to LLO pore formation. We found that loss of clathrin-independent endocytic pathways negatively influenced the efficiency of membrane repair. However, we did not detect any increased activity of these pathways, or co-localisation with the toxin or markers of membrane repair, suggesting that they were not directly involved in removal of LLO pores from the plasma membrane. In fact, markers of clathrin-independent carriers (CLICs) were rapidly disassembled in the acute phase of membrane damage due to Ca2+ influx, followed by a reassembly about 2 min after pore formation. We propose that these endocytic mechanisms might influence membrane repair by regulating the plasma membrane composition and tension, but not via direct internalisation of LLO pores.

  • 37.
    Nygård Skalman, Lars
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Holst, Mikkel Roland
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Endocytic response to the pore-forming toxin listeriolysin OManuscript (preprint) (Other academic)
  • 38.
    Olofsson, Annelie
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nygård Skalman, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Obi, Ikenna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Arnqvist, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Uptake of Helicobacter pylori vesicles is facilitated by clathrin-dependent and clathrin-independent endocytic pathways2014In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 5, no 3, p. e00979-14-Article in journal (Refereed)
    Abstract [en]

    UNLABELLED: Bacteria shed a diverse set of outer membrane vesicles that function as transport vehicles to deliver effector molecules and virulence factors to host cells. Helicobacter pylori is a gastric pathogen that infects half of the world's population, and in some individuals the infection progresses into peptic ulcer disease or gastric cancer. Here we report that intact vesicles from H. pylori are internalized by clathrin-dependent endocytosis and further dynamin-dependent processes, as well as in a cholesterol-sensitive manner. We analyzed the uptake of H. pylori vesicles by gastric epithelial cells using a method that we refer to as quantification of internalized substances (qIS). The qIS assay is based on a near-infrared dye with a cleavable linker that enables the specific quantification of internalized substances after exposure to reducing conditions. Both chemical inhibition and RNA interference in combination with the qIS assay showed that H. pylori vesicles enter gastric epithelial cells via both clathrin-mediated endocytosis and additional endocytic processes that are dependent on dynamin. Confocal microscopy revealed that H. pylori vesicles colocalized with clathrin and dynamin II and with markers of subsequent endosomal and lysosomal trafficking. Interestingly, however, knockdown of components required for caveolae had no significant effect on internalization and knockdown of components required for clathrin-independent carrier (CLIC) endocytosis increased internalization of H. pylori vesicles. Furthermore, uptake of vesicles by both clathrin-dependent and -independent pathways was sensitive to depletion, but not sequestering, of cholesterol in the host cell membrane suggesting that membrane fluidity influences the efficiency of H. pylori vesicle uptake.

    IMPORTANCE: Bacterial vesicles act as long-distance tools to deliver toxins and effector molecules to host cells. Vesicles can cause a variety of host cell responses via cell surface-induced cell signaling or internalization. Vesicles of diverse bacterial species enter host cells via different endocytic pathways or via membrane fusion. With the combination of a fluorescence-based quantification assay that quantifies internalized vesicles in a large number of cells and either chemical inhibition or RNA interference, we show that clathrin-mediated endocytosis is the major pathway for uptake of Helicobacter pylori vesicles and that lipid microdomains of the host cell membrane affect uptake of vesicles via clathrin-independent pathways. Our results provide important insights about membrane fluidity and its important role in the complex process that directs the H. pylori vesicle to a specific endocytic pathway. Understanding the mechanisms that operate in vesicle-host interactions is important to fully recognize the impact of vesicles in pathogenesis.

  • 39. Parton, Robert G.
    et al.
    del Pozo, Miguel A.
    Vassilopoulos, Stephane
    Nabi, Ivan R.
    Le Lay, Soazig
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Kenworthy, Anne K.
    Camus, Anne
    Blouin, Cedric M.
    Sessa, William C.
    Lamaze, Christophe
    Caveolae: The FAQs2019In: Traffic: the International Journal of Intracellular Transport, ISSN 1398-9219, E-ISSN 1600-0854Article, review/survey (Refereed)
    Abstract [en]

    Caveolae are an abundant, but enigmatic, plasma membrane feature of vertebrate cells. In this brief commentary, the authors attempt to answer some key questions related to the formation and function of caveolae based on round‐table discussions at the first EMBO Workshop on Caveolae held in France in May 2019.

  • 40. Pylypenko, Olena
    et al.
    Ignatev, Alexander
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rasmuson, Erika
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Carlsson, Sven R
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rak, Alexey
    A combinatorial approach to crystallization of PX-BAR unit of the human Sorting Nexin 9.2008In: Journal of Structural Biology, ISSN 1047-8477, Vol. 162, no 2, p. 356-360Article in journal (Refereed)
  • 41. Pylypenko, Olena
    et al.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rasmuson, Erika
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Carlsson, Sven R
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rak, Alexey
    The PX-BAR membrane-remodeling unit of sorting nexin 9.2007In: EMBO Journal, ISSN 1460-2075, Vol. 26, no 22, p. 4788-800Article in journal (Refereed)
  • 42.
    Rompikuntal, Pramod K.
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Vdovikova, Svitlana
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Duperthuy, Marylise
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Johnson, Tanya L.
    Åhlund, Monika
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Oscarsson, Jan
    Umeå University, Faculty of Medicine, Department of Odontology.
    Sandkvist, Maria
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Outer Membrane Vesicle-Mediated Export of Processed PrtV Protease from Vibrio cholerae2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 7, article id e0134098Article in journal (Refereed)
    Abstract [en]

    Background Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during normal growth. OMVs carry different biologically active toxins and enzymes into the surrounding environment. We suggest that OMVs may therefore be able to transport bacterial proteases into the target host cells. We present here an analysis of the Vibrio cholerae OMV-associated protease PrtV. Methodology/Principal Findings In this study, we demonstrated that PrtV was secreted from the wild type V. cholerae strain C6706 via the type II secretion system in association with OMVs. By immunoblotting and electron microscopic analysis using immunogold labeling, the association of PrtV with OMVs was examined. We demonstrated that OMV-associated PrtV was biologically active by showing altered morphology and detachment of cells when the human ileocecum carcinoma (HCT8) cells were treated with OMVs from the wild type V. cholerae strain C6706 whereas cells treated with OMVs from the prtV isogenic mutant showed no morphological changes. Furthermore, OMV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37. Conclusion/Significance Our findings suggest that OMVs released from V. cholerae can deliver a processed, biologically active form of PrtV that contributes to bacterial interactions with target host cells.

  • 43.
    Rompikuntal, Pramod Kumar
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Åhlund, Monika
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lindmark, Barbro
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Johnson, Tanya L
    Department of Microbiology and Immunology, University of Michigan Medical School..
    Sandkvist, Maria
    Department of Microbiology and Immunology, University of Michigan Medical School..
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Outer membrane vesicle-mediated export of PrtV protease from Vibrio choleraeManuscript (preprint) (Other academic)
    Abstract [en]

    Background: Gram-negative bacteria release large amounts of outer membrane vesicles (OMVs) during normal growth. OMVs from pathogenic bacteria are known to carry different biologically active toxins and enzymes into the surrounding environment. We hypothesized that OMVs may therefore be able to mediate the transport of bacterial products into host cells. We present here an analysis of the V. cholerae OMV-associated virulence factor PrtV. 

    Methodology/Principal Findings: We observed that PrtV, a M6 family, zinc-binding metalloprotease, is secreted from V. cholerae wild type strain C6706 into the culture supernatant in association with OMVs. The association of PrtV with OMVs was determined by immunoblotting and electron microscopy using immunogold labeling. In addition, we observed that the PKD-domain(s) of PrtV has a role in the protein’s association with OMVs. We also demonstrated that OMV-associated PrtV was biologically active because HCT8 cells treated with OMVs from the wild type V. cholerae strain C6706 exhibited altered morphology, whereas cells treated with OMVs from the prtV isogenic mutant showed no morphological changes. Moreover, our data suggest that OMV-associated PrtV might be transported into target eukaryotic cells by a vesicle fusion mechanism in association with lipid raft microdomains in the plasma membrane.

    Conclusion/Significance: Our findings suggest that OMVs can deliver biologically active PrtV into target host cells.

  • 44. Shah, Claudio
    et al.
    Hegde, Balachandra G
    Morén, Björn
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Behrmann, Elmar
    Mielke, Thorsten
    Moenke, Gregor
    Spahn, Christian MT
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Daumke, Oliver
    Langen, Ralf
    Structural insights into membrane interaction and caveolar targeting of dynamin-like EHD22014In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 22, no 3, p. 409-420Article in journal (Refereed)
    Abstract [en]

    The dynamin-related Eps15-homology domain-containing protein 2 (EHD2) is a membrane-remodeling ATPase that regulates the dynamics of caveolae. Here, we established an electron paramagnetic resonance (EPR) approach to characterize structural features of membrane-bound EHD2. We show that residues at the tip of the helical domain can insert into the membrane and may create membrane curvature by a wedging mechanism. Using EPR and X-ray crystallography, we found that the N terminus is folded into a hydrophobic pocket of the GTPase domain in solution and can be released into the membrane. Cryoelectron microscopy demonstrated that the N terminus is not essential for oligomerization of EHD2 into a membrane-anchored scaffold. Instead, we found a function of the N terminus in regulating targeting and stable association of EHD2 to caveolae. Our data uncover an unexpected, membrane-induced regulatory switch in EHD2 and demonstrate the versatility of EPR to study structure and function of dynamin superfamily proteins.

  • 45.
    Storm, Rickard J
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Persson, David B
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Skalman, Lars Nygård
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Frängsmyr, Lars
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Lindström, Mona
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Rankin, Greg
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Domellöf, Fatima Pedrosa
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Arnberg, Niklas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Human Adenovirus Type 37 Uses αVβ1 and α3β1 Integrins for Infection of Human Corneal Cells2017In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 91, no 5, article id e02019-16Article in journal (Refereed)
    Abstract [en]

    Epidemic keratoconjunctivitis (EKC) is a severe, contagious ocular disease that affects 20 to 40 million individuals worldwide every year. EKC is mainly caused by six types of human adenovirus (HAdV): HAdV-8, -19, -37, -53, -54, and -56. Of these, HAdV-8, -19, and -37 use sialic acid-containing glycans as cellular receptors. αVβ3, αVβ5, and a few additional integrins facilitate entry and endosomal release of other HAdVs. With the exception of a few biochemical analyses indicating that HAdV-37 can interact physically with αVβ5, little is known about the integrins used by EKC-causing HAdVs. Here, we investigated the overall integrin expression on human corneal cells and found expression of α2, α3, α6, αV, β1, and β4 subunits in human corneal in situ epithelium and/or in a human corneal epithelial (HCE) cell line but no or less accessible expression of α4, α5, β3, or β5. We also identified the integrins used by HAdV-37 through a series of binding and infection competition experiments and different biochemical approaches. Together, our data suggest that HAdV-37 uses αVβ1 and α3β1 integrins for infection of human corneal epithelial cells. Furthermore, to confirm the relevance of these integrins in the HAdV-37 life cycle, we developed a corneal multilayer tissue system and found that HAdV-37 infection correlated well with the patterns of αV, α3, and β1 integrin expression. These results provide further insight into the tropism and pathogenesis of EKC-causing HAdVs and may be of importance for future development of new antiviral drugs.IMPORTANCE Keratitis is a hallmark of EKC, which is caused by six HAdV types (HAdV-8, -19, -37, -53, -54, and -56). HAdV-37 and some other HAdV types interact with integrin αVβ5 in order to enter nonocular human cells. In this study, we found that αVβ5 is not expressed on human corneal epithelial cells, thus proposing other host factors mediate corneal infection. Here, we first characterized integrin expression patterns on corneal tissue and corneal cells. Among the integrins identified, competition binding and infection experiments and biochemical assays pointed out αVβ1 and α3β1 to be of importance for HAdV-37 infection of corneal tissue. In the absence of a good animal model for EKC-causing HAdVs, we also developed an in vitro system with multilayer HCE cells and confirmed the relevance of the suggested integrins during HAdV-37 infection.

  • 46.
    Vdovikova, Svitlana
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Luhr, Morten
    Szalai, Paula
    Skalman, Lars Nygård
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Francis, Monika K.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Engedal, Nikolai
    Johansson, Jörgen
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wai, Sun N.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    A Novel Role of Listeria monocytogenes Membrane Vesicles in Inhibition of Autophagy and Cell Death2017In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 7, article id 154Article in journal (Refereed)
    Abstract [en]

    Bacterial membrane vesicle (MV) production has been mainly studied in Gram-negative species. In this study, we show that Listeria monocytogenes, a Gram-positive pathogen that causes the food-borne illness listeriosis, produces MVs both in vitro and in vivo. We found that a major virulence factor, the pore-forming hemolysin listeriolysin O (LLO), is tightly associated with the MVs, where it resides in an oxidized, inactive state. Previous studies have shown that LLO may induce cell death and autophagy. To monitor possible effects of LLO and MVs on autophagy, we performed assays for LC3 lipidation and LDH sequestration as well as analysis by confocal microscopy of HEK293 cells expressing GFP-LC3. The results revealed that MVs alone did not affect autophagy whereas they effectively abrogated autophagy induced by pure LLO or by another pore-forming toxin from Vibrio cholerae, VCC. Moreover, Listeria monocytogenes MVs significantly decreased Torin1-stimulated macroautophagy. In addition, MVs protected against necrosis of HEK293 cells caused by the lytic action of LLO. We explored the mechanisms of LLO-induced autophagy and cell death and demonstrated that the protective effect of MVs involves an inhibition of LLO-induced pore formation resulting in inhibition of autophagy and the lytic action on eukaryotic cells. Further, we determined that these MVs help bacteria to survive inside eukaryotic cells (mouse embryonic fibroblasts). Taken together, these findings suggest that intracellular release of MVs from L. monocytogenes may represent a bacterial strategy to survive inside host cells, by its control of LLO activity and by avoidance of destruction from the autophagy system during infection.

  • 47.
    Vidal-Quadras, Maite
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Holst, Mikkel R.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Francis, Monika K.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Larsson, Elin
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hachimi, Mariam
    Yau, Wai-Lok
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Peranen, Johan
    Martin-Belmonte, Fernando
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Endocytic turnover of Rab8 controls cell polarization2017In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 130, no 6, p. 1147-1157Article in journal (Refereed)
    Abstract [en]

    Adaptation of cell shape and polarization through the formation and retraction of cellular protrusions requires balancing of endocytosis and exocytosis combined with fine-tuning of the local activity of small GTPases like Rab8. Here, we show that endocytic turnover of the plasma membrane at protrusions is directly coupled to surface removal and inactivation of Rab8. Removal is induced by reduced membrane tension and mediated by the GTPase regulator associated with focal adhesion kinase-1 (GRAF1, also known as ARHGAP26), a regulator of clathrin-independent endocytosis. GRAF1-depleted cells were deficient in multi-directional spreading and displayed elevated levels of GTP-loaded Rab8, which was accumulated at the tips of static protrusions. Furthermore, GRAF1 depletion impaired lumen formation and spindle orientation in a 3D cell culture system, indicating that GRAF1 activity regulates polarity establishment. Our data suggest that GRAF1-mediated removal of Rab8 from the cell surface restricts its activity during protrusion formation, thereby facilitating dynamic adjustment of the polarity axis.

  • 48.
    Vidal-Quadras, Maite
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Holst, Mikkel R.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Francis, Monika K.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Peränen, Johan
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Endocytic turnover of Rab8 controls cell polarisationManuscript (preprint) (Other academic)
  • 49.
    Vonderstein, Kirstin
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Nilsson, Emma
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Hubel, Philipp
    Nygård Skalman, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Upadhyay, Arunkumar
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Pasto, Jenny
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Pichlmair, Andreas
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Överby, Anna K.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Viperin targets flavivirus virulence by inducing assembly of non-infectious capsid particles2018In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 92, no 1, article id e01751-17Article in journal (Refereed)
    Abstract [en]

    Efficient antiviral immunity requires interference with virus replication at multiple layers targeting diverse steps in the viral life cycle. Here we describe a novel flavivirus inhibition mechanism that results in interferon-mediated obstruction of tick-borne encephalitis virus particle assembly, and involves release of malfunctional membrane associated capsid (C) particles. This mechanism is controlled by the activity of the interferon-induced protein viperin, a broad spectrum antiviral interferon stimulated gene. Through analysis of the viperin-interactome, we identified the Golgi Brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1), as the cellular protein targeted by viperin. Viperin-induced antiviral activity as well as C-particle release was stimulated by GBF1 inhibition and knock down, and reduced by elevated levels of GBF1. Our results suggest that viperin targets flavivirus virulence by inducing the secretion of unproductive non-infectious virus particles, by a GBF1-dependent mechanism. This yet undescribed antiviral mechanism allows potential therapeutic intervention.Importance The interferon response can target viral infection on almost every level, however, very little is known about interference of flavivirus assembly. Here we show that interferon, through the action of viperin, can disturb assembly of tick-borne encephalitis virus. The viperin protein is highly induced after viral infection and exhibit broad-spectrum antiviral activity. However, the mechanism of action is still elusive and appear to vary between the different viruses, indicating that cellular targets utilized by several viruses might be involved. In this study we show that viperin induce capsid particle release by interacting and inhibiting the function of the cellular protein Golgi Brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1). GBF1 is a key protein in the cellular secretory pathway and essential in the life cycle of many viruses, also targeted by viperin, implicating GBF1 as a novel putative drug target.

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