Recent Submissions

  • The interferon-stimulated gene product oligoadenylate synthetase-like protein enhances replication of Kaposi's sarcoma-associated herpesvirus (KSHV) and interacts with the KSHV ORF20 protein.

    Bussey, Kendra A; Lau, Ulrike; Schumann, Sophie; Gallo, Antonio; Osbelt, Lisa; Stempel, Markus; Arnold, Christine; Wissing, Josef; Gad, Hans Henrik; Hartmann, Rune; Brune, Wolfram; Jänsch, Lothar; Whitehouse, Adrian; Brinkmann, Melanie M; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-03)
    Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the few oncogenic human viruses known to date. Its large genome encodes more than 85 proteins and includes both unique viral proteins as well as proteins conserved amongst herpesviruses. KSHV ORF20 is a member of the herpesviral core UL24 family, but the function of ORF20 and its role in the viral life cycle is not well understood. ORF20 encodes three largely uncharacterized isoforms, which we found were localized predominantly in the nuclei and nucleoli. Quantitative affinity purification coupled to mass spectrometry (q-AP-MS) identified numerous specific interacting partners of ORF20, including ribosomal proteins and the interferon-stimulated gene product (ISG) oligoadenylate synthetase-like protein (OASL). Both endogenous and transiently transfected OASL co-immunoprecipitated with ORF20, and this interaction was conserved among all ORF20 isoforms and multiple ORF20 homologs of the UL24 family in other herpesviruses. Characterization of OASL interacting partners by q-AP-MS identified a very similar interactome to that of ORF20. Both ORF20 and OASL copurified with 40S and 60S ribosomal subunits, and when they were co-expressed, they associated with polysomes. Although ORF20 did not have a global effect on translation, ORF20 enhanced RIG-I induced expression of endogenous OASL in an IRF3-dependent but IFNAR-independent manner. OASL has been characterized as an ISG with antiviral activity against some viruses, but its role for gammaherpesviruses was unknown. We show that OASL and ORF20 mRNA expression were induced early after reactivation of latently infected HuARLT-rKSHV.219 cells. Intriguingly, we found that OASL enhanced infection of KSHV. During infection with a KSHV ORF20stop mutant, however, OASL-dependent enhancement of infectivity was lost. Our data have characterized the interaction of ORF20 with OASL and suggest ORF20 usurps the function of OASL to benefit KSHV infection.
  • Diverse functions of myosin VI elucidated by an isoform-specific α-helix domain.

    Wollscheid, Hans-Peter; Biancospino, Matteo; He, Fahu; Magistrati, Elisa; Molteni, Erika; Lupia, Michela; Soffientini, Paolo; Rottner, Klemens; Cavallaro, Ugo; Pozzoli, Uberto; Mapelli, Marina; Walters, Kylie J; Polo, Simona; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-04)
    Myosin VI functions in endocytosis and cell motility. Alternative splicing of myosin VI mRNA generates two distinct isoform types, myosin VI(short) and myosin VI(long), which differ in the C-terminal region. Their physiological and pathological roles remain unknown. Here we identified an isoform-specific regulatory helix, named the α2-linker, that defines specific conformations and hence determines the target selectivity of human myosin VI. The presence of the α2-linker structurally defines a new clathrin-binding domain that is unique to myosin VI(long) and masks the known RRL interaction motif. This finding is relevant to ovarian cancer, in which alternative myosin VI splicing is aberrantly regulated, and exon skipping dictates cell addiction to myosin VI(short) in tumor-cell migration. The RRL interactor optineurin contributes to this process by selectively binding myosin VI(short). Thus, the α2-linker acts like a molecular switch that assigns myosin VI to distinct endocytic (myosin VI(long)) or migratory (myosin VI(short)) functional roles.
  • Diversified actin protrusions promote environmental exploration but are dispensable for locomotion of leukocytes.

    Leithner, Alexander; Eichner, Alexander; Müller, Jan; Reversat, Anne; Brown, Markus; Schwarz, Jan; Merrin, Jack; de Gorter, David J J; Schur, Florian; Bayerl, Jonathan; de Vries, Ingrid; Wieser, Stefan; Hauschild, Robert; Lai, Frank P L; Moser, Markus; Kerjaschki, Dontscho; Rottner, Klemens; Small, J Victor; Stradal, Theresia E B; Sixt, Michael; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH, Feodor-Lynnen-Str.7, 30625 Hannover, Germany. (2016-11)
    Most migrating cells extrude their front by the force of actin polymerization. Polymerization requires an initial nucleation step, which is mediated by factors establishing either parallel filaments in the case of filopodia or branched filaments that form the branched lamellipodial network. Branches are considered essential for regular cell motility and are initiated by the Arp2/3 complex, which in turn is activated by nucleation-promoting factors of the WASP and WAVE families. Here we employed rapid amoeboid crawling leukocytes and found that deletion of the WAVE complex eliminated actin branching and thus lamellipodia formation. The cells were left with parallel filaments at the leading edge, which translated, depending on the differentiation status of the cell, into a unipolar pointed cell shape or cells with multiple filopodia. Remarkably, unipolar cells migrated with increased speed and enormous directional persistence, while they were unable to turn towards chemotactic gradients. Cells with multiple filopodia retained chemotactic activity but their migration was progressively impaired with increasing geometrical complexity of the extracellular environment. These findings establish that diversified leading edge protrusions serve as explorative structures while they slow down actual locomotion.
  • A Genome-Wide siRNA Screen Implicates Spire1/2 in SipA-Driven Salmonella Typhimurium Host Cell Invasion.

    Andritschke, Daniel; Dilling, Sabrina; Emmenlauer, Mario; Welz, Tobias; Schmich, Fabian; Misselwitz, Benjamin; Rämö, Pauli; Rottner, Klemens; Kerkhoff, Eugen; Wada, Teiji; Penninger, Josef M; Beerenwinkel, Niko; Horvath, Peter; Dehio, Christoph; Hardt, Wolf-Dietrich; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016)
    Salmonella Typhimurium (S. Tm) is a leading cause of diarrhea. The disease is triggered by pathogen invasion into the gut epithelium. Invasion is attributed to the SPI-1 type 3 secretion system (T1). T1 injects effector proteins into epithelial cells and thereby elicits rearrangements of the host cellular actin cytoskeleton and pathogen invasion. The T1 effector proteins SopE, SopB, SopE2 and SipA are contributing to this. However, the host cell factors contributing to invasion are still not completely understood. To address this question comprehensively, we used Hela tissue culture cells, a genome-wide siRNA library, a modified gentamicin protection assay and S. TmSipA, a sopBsopE2sopE mutant which strongly relies on the T1 effector protein SipA to invade host cells. We found that S. TmSipA invasion does not elicit membrane ruffles, nor promote the entry of non-invasive bacteria "in trans". However, SipA-mediated infection involved the SPIRE family of actin nucleators, besides well-established host cell factors (WRC, ARP2/3, RhoGTPases, COPI). Stage-specific follow-up assays and knockout fibroblasts indicated that SPIRE1 and SPIRE2 are involved in different steps of the S. Tm infection process. Whereas SPIRE1 interferes with bacterial binding, SPIRE2 influences intracellular replication of S. Tm. Hence, these two proteins might fulfill non-redundant functions in the pathogen-host interaction. The lack of co-localization hints to a short, direct interaction between S. Tm and SPIRE proteins or to an indirect effect.
  • Kindlin-2 recruits paxillin and Arp2/3 to promote membrane protrusions during initial cell spreading.

    Böttcher, Ralph T; Veelders, Maik; Rombaut, Pascaline; Faix, Jan; Theodosiou, Marina; Stradal, Theresia E B; Rottner, Klemens; Zent, Roy; Herzog, Franz; Fässler, Reinhard; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-09-14)
    Cell spreading requires the coupling of actin-driven membrane protrusion and integrin-mediated adhesion to the extracellular matrix. The integrin-activating adaptor protein kindlin-2 plays a central role for cell adhesion and membrane protrusion by directly binding and recruiting paxillin to nascent adhesions. Here, we report that kindlin-2 has a dual role during initial cell spreading: it binds paxillin via the pleckstrin homology and F0 domains to activate Rac1, and it directly associates with the Arp2/3 complex to induce Rac1-mediated membrane protrusions. Consistently, abrogation of kindlin-2 binding to Arp2/3 impairs lamellipodia formation and cell spreading. Our findings identify kindlin-2 as a key protein that couples cell adhesion by activating integrins and the induction of membrane protrusions by activating Rac1 and supplying Rac1 with the Arp2/3 complex.
  • Actin assembly mechanisms at a glance.

    Rottner, Klemens; Faix, Jan; Bogdan, Sven; Linder, Stefan; Kerkhoff, Eugen; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-10-15)
    The actin cytoskeleton and associated motor proteins provide the driving forces for establishing the astonishing morphological diversity and dynamics of mammalian cells. Aside from functions in protruding and contracting cell membranes for motility, differentiation or cell division, the actin cytoskeleton provides forces to shape and move intracellular membranes of organelles and vesicles. To establish the many different actin assembly functions required in time and space, actin nucleators are targeted to specific subcellular compartments, thereby restricting the generation of specific actin filament structures to those sites. Recent research has revealed that targeting and activation of actin filament nucleators, elongators and myosin motors are tightly coordinated by conserved protein complexes to orchestrate force generation. In this Cell Science at a Glance article and the accompanying poster, we summarize and discuss the current knowledge on the corresponding protein complexes and their modes of action in actin nucleation, elongation and force generation.
  • FMNL2 and -3 regulate Golgi architecture and anterograde transport downstream of Cdc42.

    Kage, Frieda; Steffen, Anika; Ellinger, Adolf; Ranftler, Carmen; Gehre, Christian; Brakebusch, Cord; Pavelka, Margit; Stradal, Theresia; Rottner, Klemens; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-08-29)
    The Rho-family small GTPase Cdc42 localizes at plasma membrane and Golgi complex and aside from protrusion and migration operates in vesicle trafficking, endo- and exocytosis as well as establishment and/or maintenance of cell polarity. The formin family members FMNL2 and -3 are actin assembly factors established to regulate cell edge protrusion during migration and invasion. Here we report these formins to additionally accumulate and function at the Golgi apparatus. As opposed to lamellipodia, Golgi targeting of these proteins required both their N-terminal myristoylation and the interaction with Cdc42. Moreover, Golgi association of FMNL2 or -3 induced a phalloidin-detectable actin meshwork around the Golgi. Importantly, functional interference with FMNL2/3 formins by RNAi or CRISPR/Cas9-mediated gene deletion invariably induced Golgi fragmentation in different cell lines. Furthermore, absence of these proteins led to enlargement of endosomes as well as defective maturation and/or sorting into late endosomes and lysosomes. In line with Cdc42 - recently established to regulate anterograde transport through the Golgi by cargo sorting and carrier formation - FMNL2/3 depletion also affected anterograde trafficking of VSV-G from the Golgi to the plasma membrane. Our data thus link FMNL2/3 formins to actin assembly-dependent functions of Cdc42 in anterograde transport through the Golgi apparatus.
  • A highly conserved redox-active Mx(2)CWx(6)R motif regulates Zap70 stability and activity.

    Thurm, Christoph; Poltorak, Mateusz P; Reimer, Elisa; Brinkmann, Melanie M; Leichert, Lars; Schraven, Burkhart; Simeoni, Luca; Helmholtz Centre of infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-05-09)
    ζ-associated protein of 70 kDa (Zap70) is crucial for T-cell receptor (TCR) signaling. Loss of Zap70 in both humans and mice results in severe immunodeficiency. On the other hand, the expression of Zap70 in B-cell malignancies correlates with the severity of the disease. Because of its role in immune-related disorders, Zap70 has become a therapeutic target for the treatment of human diseases. It is well-established that the activity/expression of Zap70 is regulated by post-translational modifications of crucial amino acids including the phosphorylation of tyrosines and the ubiquitination of lysines. Here, we have investigated whether also oxidation of cysteine residues regulates Zap70 functions. We have identified C575 as a major sulfenylation site of Zap70. A C575A substitution results in protein instability, reduced activity, and increased dependency on the Hsp90/Cdc37 chaperone system. Indeed, Cdc37 overexpression reconstituted partially the expression but fully the function of Zap70C575A. C575 lies within a Mx(2)CWx(6)R motif which is highly conserved among almost all human tyrosine kinases. Mutation of any of the conserved amino acids, but not of a non-conserved residue preceding the cysteine, also results in Zap70 instability. Collectively, we have identified a new redox-active motif which is crucial for the regulation of Zap70 stability/activity. We believe that this motif has the potential to become a novel target for the development of therapeutic tools to modulate the expression/activity of kinases.
  • The Mouse Cytomegalovirus Gene m42 Targets Surface Expression of the Protein Tyrosine Phosphatase CD45 in Infected Macrophages.

    Thiel, Nadine; Keyser, Kirsten A; Lemmermann, Niels A W; Oduro, Jennifer D; Wagner, Karen; Elsner, Carina; Halenius, Anne; Lenac Roviš, Tihana; Brinkmann, Melanie M; Jonjić, Stipan; Cicin-Sain, Luka; Messerle, Martin; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-12)
    The receptor-like protein tyrosine phosphatase CD45 is expressed on the surface of cells of hematopoietic origin and has a pivotal role for the function of these cells in the immune response. Here we report that following infection of macrophages with mouse cytomegalovirus (MCMV) the cell surface expression of CD45 is drastically diminished. Screening of a set of MCMV deletion mutants allowed us to identify the viral gene m42 of being responsible for CD45 down-modulation. Moreover, expression of m42 independent of viral infection upon retroviral transduction of the RAW264.7 macrophage cell line led to comparable regulation of CD45 expression. In immunocompetent mice infected with an m42 deletion mutant lower viral titers were observed in all tissues examined when compared to wildtype MCMV, indicating an important role of m42 for viral replication in vivo. The m42 gene product was identified as an 18 kDa protein expressed with early kinetics and is predicted to be a tail-anchored membrane protein. Tracking of surface-resident CD45 molecules revealed that m42 induces internalization and degradation of CD45. The observation that the amounts of the E3 ubiquitin ligases Itch and Nedd4 were diminished in cells expressing m42 and that disruption of a PY motif in the N-terminal part of m42 resulted in loss of function, suggest that m42 acts as an activator or adaptor for these Nedd4-like ubiquitin ligases, which mark CD45 for lysosomal degradation. In conclusion, the down-modulation of CD45 expression in MCMV-infected myeloid cells represents a novel pathway of virus-host interaction.
  • The murine cytomegalovirus M35 protein antagonizes type I IFN induction downstream of pattern recognition receptors by targeting NF-κB mediated transcription.

    Chan, Baca; Gonçalves Magalhães, Vladimir; Lemmermann, Niels A W; Juranić Lisnić, Vanda; Stempel, Markus; Bussey, Kendra A; Reimer, Elisa; Podlech, Jürgen; Lienenklaus, Stefan; Reddehase, Matthias J; Jonjić, Stipan; Brinkmann, Melanie M; Helmholtz Centre for infection research, Inhoffenstr. 7., 38124 Braunschweig, Germany. (2017-05)
    The type I interferon (IFN) response is imperative for the establishment of the early antiviral immune response. Here we report the identification of the first type I IFN antagonist encoded by murine cytomegalovirus (MCMV) that shuts down signaling following pattern recognition receptor (PRR) sensing. Screening of an MCMV open reading frame (ORF) library identified M35 as a novel and strong negative modulator of IFNβ promoter induction following activation of both RNA and DNA cytoplasmic PRR. Additionally, M35 inhibits the proinflammatory cytokine response downstream of Toll-like receptors (TLR). Using a series of luciferase-based reporters with specific transcription factor binding sites, we determined that M35 targets NF-κB-, but not IRF-mediated, transcription. Expression of M35 upon retroviral transduction of immortalized bone marrow-derived macrophages (iBMDM) led to reduced IFNβ transcription and secretion upon activation of stimulator of IFN genes (STING)-dependent signaling. On the other hand, M35 does not antagonize interferon-stimulated gene (ISG) 56 promoter induction or ISG transcription upon exogenous stimulation of the type I IFN receptor (IFNAR). M35 is present in the viral particle and, upon MCMV infection of fibroblasts, is immediately shuttled to the nucleus where it exerts its immunomodulatory effects. Deletion of M35 from the MCMV genome and hence from the viral particle resulted in elevated type I IFN transcription and secretion in vitro and in vivo. In the absence of M35, lower viral titers are observed during acute infection of the host, and productive infection in the salivary glands was not detected. In conclusion, the M35 protein is released by MCMV immediately upon infection in order to deftly inhibit the antiviral type I IFN response by targeting NF-κB-mediated transcription. The identification of this novel viral protein reinforces the importance of timely countermeasures in the complex relationship between virus and host.
  • Coordination by Cdc42 of Actin, Contractility, and Adhesion for Melanoblast Movement in Mouse Skin.

    Woodham, Emma F; Paul, Nikki R; Tyrrell, Benjamin; Spence, Heather J; Swaminathan, Karthic; Scribner, Michelle R; Giampazolias, Evangelos; Hedley, Ann; Clark, William; Kage, Frieda; Marston, Daniel J; Hahn, Klaus M; Tait, Stephen W G; Larue, Lionel; Brakebusch, Cord H; Insall, Robert H; Machesky, Laura M; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-03-06)
    The individual molecular pathways downstream of Cdc42, Rac, and Rho GTPases are well documented, but we know surprisingly little about how these pathways are coordinated when cells move in a complex environment in vivo. In the developing embryo, melanoblasts originating from the neural crest must traverse the dermis to reach the epidermis of the skin and hair follicles. We previously established that Rac1 signals via Scar/WAVE and Arp2/3 to effect pseudopod extension and migration of melanoblasts in skin. Here we show that RhoA is redundant in the melanocyte lineage but that Cdc42 coordinates multiple motility systems independent of Rac1. Similar to Rac1 knockouts, Cdc42 null mice displayed a severe loss of pigmentation, and melanoblasts showed cell-cycle progression, migration, and cytokinesis defects. However, unlike Rac1 knockouts, Cdc42 null melanoblasts were elongated and displayed large, bulky pseudopods with dynamic actin bursts. Despite assuming an elongated shape usually associated with fast mesenchymal motility, Cdc42 knockout melanoblasts migrated slowly and inefficiently in the epidermis, with nearly static pseudopods. Although much of the basic actin machinery was intact, Cdc42 null cells lacked the ability to polarize their Golgi and coordinate motility systems for efficient movement. Loss of Cdc42 de-coupled three main systems: actin assembly via the formin FMNL2 and Arp2/3, active myosin-II localization, and integrin-based adhesion dynamics.
  • FMNL formins boost lamellipodial force generation.

    Kage, Frieda; Winterhoff, Moritz; Dimchev, Vanessa; Mueller, Jan; Thalheim, Tobias; Freise, Anika; Brühmann, Stefan; Kollasser, Jana; Block, Jennifer; Dimchev, Georgi; Geyer, Matthias; Schnittler, Hans-Joachim; Brakebusch, Cord; Stradal, Theresia E B; Carlier, Marie-France; Sixt, Michael; Käs, Josef; Faix, Jan; Rottner, Klemens; Helmholtz Centre for infection research, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-03-22)
    Migration frequently involves Rac-mediated protrusion of lamellipodia, formed by Arp2/3 complex-dependent branching thought to be crucial for force generation and stability of these networks. The formins FMNL2 and FMNL3 are Cdc42 effectors targeting to the lamellipodium tip and shown here to nucleate and elongate actin filaments with complementary activities in vitro. In migrating B16-F1 melanoma cells, both formins contribute to the velocity of lamellipodium protrusion. Loss of FMNL2/3 function in melanoma cells and fibroblasts reduces lamellipodial width, actin filament density and -bundling, without changing patterns of Arp2/3 complex incorporation. Strikingly, in melanoma cells, FMNL2/3 gene inactivation almost completely abolishes protrusion forces exerted by lamellipodia and modifies their ultrastructural organization. Consistently, CRISPR/Cas-mediated depletion of FMNL2/3 in fibroblasts reduces both migration and capability of cells to move against viscous media. Together, we conclude that force generation in lamellipodia strongly depends on FMNL formin activity, operating in addition to Arp2/3 complex-dependent filament branching.
  • Loss of cortactin causes endothelial barrier dysfunction via disturbed adrenomedullin secretion and actomyosin contractility.

    García Ponce, Alexander; Citalán Madrid, Alí F; Vargas Robles, Hilda; Chánez Paredes, Sandra; Nava, Porfirio; Betanzos, Abigail; Zarbock, Alexander; Rottner, Klemens; Vestweber, Dietmar; Schnoor, Michael; Department for Molecular Biomedicine, Center of Research and Advanced Studies (CINVESTAV-IPN), 07360 Mexico-City, Mexico. (2016)
    Changes in vascular permeability occur during inflammation and the actin cytoskeleton plays a crucial role in regulating endothelial cell contacts and permeability. We demonstrated recently that the actin-binding protein cortactin regulates vascular permeability via Rap1. However, it is unknown if the actin cytoskeleton contributes to increased vascular permeability without cortactin. As we consistently observed more actin fibres in cortactin-depleted endothelial cells, we hypothesised that cortactin depletion results in increased stress fibre contractility and endothelial barrier destabilisation. Analysing the contractile machinery, we found increased ROCK1 protein levels in cortactin-depleted endothelium. Concomitantly, myosin light chain phosphorylation was increased while cofilin, mDia and ERM were unaffected. Secretion of the barrier-stabilising hormone adrenomedullin, which activates Rap1 and counteracts actomyosin contractility, was reduced in plasma from cortactin-deficient mice and in supernatants of cortactin-depleted endothelium. Importantly, adrenomedullin administration and ROCK1 inhibition reduced actomyosin contractility and rescued the effect on permeability provoked by cortactin deficiency in vitro and in vivo. Our data suggest a new role for cortactin in controlling actomyosin contractility with consequences for endothelial barrier integrity.
  • Perinuclear Arp2/3-driven actin polymerization enables nuclear deformation to facilitate cell migration through complex environments.

    Thiam, Hawa-Racine; Vargas, Pablo; Carpi, Nicolas; Crespo, Carolina Lage; Raab, Matthew; Terriac, Emmanuel; King, Megan C; Jacobelli, Jordan; Alberts, Arthur S; Stradal, Theresia; Lennon-Dumenil, Ana-Maria; Piel, Matthieu; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016)
    Cell migration has two opposite faces: although necessary for physiological processes such as immune responses, it can also have detrimental effects by enabling metastatic cells to invade new organs. In vivo, migration occurs in complex environments and often requires a high cellular deformability, a property limited by the cell nucleus. Here we show that dendritic cells, the sentinels of the immune system, possess a mechanism to pass through micrometric constrictions. This mechanism is based on a rapid Arp2/3-dependent actin nucleation around the nucleus that disrupts the nuclear lamina, the main structure limiting nuclear deformability. The cells' requirement for Arp2/3 to pass through constrictions can be relieved when nuclear stiffness is decreased by suppressing lamin A/C expression. We propose a new role for Arp2/3 in three-dimensional cell migration, allowing fast-moving cells such as leukocytes to rapidly and efficiently migrate through narrow gaps, a process probably important for their function.
  • How distinct Arp2/3 complex variants regulate actin filament assembly.

    Rottner, Klemens; Stradal, Theresia E B; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2015-12-23)
    The heptameric Arp2/3 complex generates branched actin filament networks that drive lamellipodium protrusion, vesicle trafficking and pathogen motility. Distinct variants of the Arp2/3 complex are now shown to have different roles in tuning actin assembly and disassembly, in concert with the prominent actin regulators cortactin and coronin.
  • The structure of FMNL2-Cdc42 yields insights into the mechanism of lamellipodia and filopodia formation.

    Kühn, Sonja; Erdmann, Constanze; Kage, Frieda; Block, Jennifer; Schwenkmezger, Lisa; Steffen, Anika; Rottner, Klemens; Geyer, Matthias; Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany. (2015)
    Formins are actin polymerization factors that elongate unbranched actin filaments at the barbed end. Rho family GTPases activate Diaphanous-related formins through the relief of an autoregulatory interaction. The crystal structures of the N-terminal domains of human FMNL1 and FMNL2 in complex with active Cdc42 show that Cdc42 mediates contacts with all five armadillo repeats of the formin with specific interactions formed by the Rho-GTPase insert helix. Mutation of three residues within Rac1 results in a gain-of-function mutation for FMNL2 binding and reconstitution of the Cdc42 phenotype in vivo. Dimerization of FMNL1 through a parallel coiled coil segment leads to formation of an umbrella-shaped structure that—together with Cdc42—spans more than 15 nm in diameter. The two interacting FMNL-Cdc42 heterodimers expose six membrane interaction motifs on a convex protein surface, the assembly of which may facilitate actin filament elongation at the leading edge of lamellipodia and filopodia.
  • The EHEC-host interactome reveals novel targets for the translocated intimin receptor.

    Blasche, Sonja; Arens, Stefan; Ceol, Arnaud; Siszler, Gabriella; Schmidt, M Alexander; Häuser, Roman; Schwarz, Frank; Wuchty, Stefan; Aloy, Patrick; Uetz, Peter; Stradal, Theresia; Koegl, Manfred; Helmholtz Centre for infection research; Inhooffenstr. 7; D-38124 Braunschweig; Germany. (2014)
    Enterohemorrhagic E. coli (EHEC) manipulate their human host through at least 39 effector proteins which hijack host processes through direct protein-protein interactions (PPIs). To identify their protein targets in the host cells, we performed yeast two-hybrid screens, allowing us to find 48 high-confidence protein-protein interactions between 15 EHEC effectors and 47 human host proteins. In comparison to other bacteria and viruses we found that EHEC effectors bind more frequently to hub proteins as well as to proteins that participate in a higher number of protein complexes. The data set includes six new interactions that involve the translocated intimin receptor (TIR), namely HPCAL1, HPCAL4, NCALD, ARRB1, PDE6D, and STK16. We compared these TIR interactions in EHEC and enteropathogenic E. coli (EPEC) and found that five interactions were conserved. Notably, the conserved interactions included those of serine/threonine kinase 16 (STK16), hippocalcin-like 1 (HPCAL1) as well as neurocalcin-delta (NCALD). These proteins co-localize with the infection sites of EPEC. Furthermore, our results suggest putative functions of poorly characterized effectors (EspJ, EspY1). In particular, we observed that EspJ is connected to the microtubule system while EspY1 appears to be involved in apoptosis/cell cycle regulation.
  • Age-dependent enterocyte invasion and microcolony formation by Salmonella.

    Zhang, Kaiyi; Dupont, Aline; Torow, Natalia; Gohde, Fredrik; Leschner, Sara; Lienenklaus, Stefan; Weiss, Siegfried; Brinkmann, Melanie M; Kühnel, Mark; Hensel, Michael; Fulde, Marcus; Hornef, Mathias W (2014-09)
    The coordinated action of a variety of virulence factors allows Salmonella enterica to invade epithelial cells and penetrate the mucosal barrier. The influence of the age-dependent maturation of the mucosal barrier for microbial pathogenesis has not been investigated. Here, we analyzed Salmonella infection of neonate mice after oral administration. In contrast to the situation in adult animals, we observed spontaneous colonization, massive invasion of enteroabsorptive cells, intraepithelial proliferation and the formation of large intraepithelial microcolonies. Mucosal translocation was dependent on enterocyte invasion in neonates in the absence of microfold (M) cells. It further resulted in potent innate immune stimulation in the absence of pronounced neutrophil-dominated pathology. Our results identify factors of age-dependent host susceptibility and provide important insight in the early steps of Salmonella infection in vivo. We also present a new small animal model amenable to genetic manipulation of the host for the analysis of the Salmonella enterocyte interaction in vivo.
  • Superior induction and maintenance of protective CD8 T cells in mice infected with mouse cytomegalovirus vector expressing RAE-1γ.

    Trsan, Tihana; Busche, Andreas; Abram, Maja; Wensveen, Felix M; Lemmermann, Niels A; Arapovic, Maja; Babic, Marina; Tomic, Adriana; Golemac, Mijo; Brinkmann, Melanie M; Jäger, Wiebke; Oxenius, Annette; Polic, Bojan; Krmpotic, Astrid; Messerle, Martin; Jonjic, Stipan; Research group viral immune modulation, Helmholtz Centre for infection research, Braunschweig, Germany (2013-10-08)
    Due to a unique pattern of CD8 T-cell response induced by cytomegaloviruses (CMVs), live attenuated CMVs are attractive candidates for vaccine vectors for a number of clinically relevant infections and tumors. NKG2D is one of the most important activating NK cell receptors that plays a role in costimulation of CD8 T cells. Here we demonstrate that the expression of CD8 T-cell epitope of Listeria monocytogenes by a recombinant mouse CMV (MCMV) expressing the NKG2D ligand retinoic acid early-inducible protein 1-gamma (RAE-1γ) dramatically enhanced the effectiveness and longevity of epitope-specific CD8 T-cell response and conferred protection against a subsequent challenge infection with Listeria monocytogenes. Unexpectedly, the attenuated growth in vivo of the CMV vector expressing RAE-1γ and its capacity to enhance specific CD8 T-cell response were preserved even in mice lacking NKG2D, implying additional immune function for RAE-1γ beyond engagement of NKG2D. Thus, vectors expressing RAE-1γ represent a promising approach in the development of CD8 T-cell-based vaccines.
  • Measurement of mouse cytomegalovirus-induced interferon-β with immortalized luciferase reporter cells.

    Scheibe, Evgenia; Lienenklaus, Stefan; May, Tobias; Magalhães, Vladimir Gonçalves; Weiss, Siegfried; Brinkmann, Melanie M; Helmholtz Centre for Infection Research, Braunschweig, Germany. (2013)
    The production of cytokines is a crucial element of the host response to viral and bacterial infections. To follow these events in vivo, transgenic mice have become a valuable tool to study cytokine production through induction of reporter genes. We describe here the generation and immortalization of cells derived from transgenic reporter mice for development of a high-throughput assay system for virus- or bacteria-induced cytokine induction. As an example we describe mouse cytomegalovirus (MCMV) infection of immortalized fibroblasts derived from mice expressing the firefly luciferase reporter downstream of the IFN-β promoter. Common methods to determine IFN-β production, including ELISA, quantitative real-time PCR (qPCR), and transient reporter assays using plasmid-based reporter constructs, have disadvantages and limitations. Transient transfections influence type I IFN responses in most cell types, and IFN-β ELISA as well as qPCR are both laborious and expensive. The method presented here is highly sensitive as well as cost-effective, and allows monitoring of a robust and dose-dependent induction of IFN-β upon virus infection in cell lysates as well as living cells.

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