Recent Submissions

  • The endosomal Toll-like receptors 7 and 9 cooperate in detection of MHV68 infection.

    Bussey, Kendra A; Murthy, Sripriya; Reimer, Elisa; Chan, Baca; Hatesuer, Bastian; Schughart, Klaus; Glaunsinger, Britt; Adler, Heiko; Brinkmann, Melanie M; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Amercan Society of Microbiology, 2018-11-14)
    Murine gammaherpesvirus 68 (MHV68) is an amenable small animal model for study of the human pathogens Epstein-Barr virus and Kaposi’s sarcoma-associated herpesvirus. Here, we have characterized the roles of the endosomal TLR escort protein UNC93B, endosomal TLR7, 9, and 13, and cell surface TLR2 in MHV68 detection. We found that the interferon α (IFNα) response of plasmacytoid dendritic cells (pDC) to MHV68 was reduced in Tlr9-/- cells compared to wildtype (WT), but not completely lost. Tlr7-/- pDC responded similarly to WT. However, we found that in Unc93b-/- pDC, as well as in Tlr7/Tlr9-/- double knockout pDC, the IFNα response to MHV68 was completely abolished. Thus, the only pattern recognition receptors contributing to the IFNα response to MHV68 in pDC are TLR7 and TLR9, but the contribution of TLR7 is masked by the presence of TLR9. To address the role of UNC93B and TLR for MHV68 infection in vivo, we infected mice with MHV68. Lytic replication of MHV68 after intravenous infection was enhanced in the lungs, spleen, and liver of UNC93B-deficient mice, in the spleen of TLR9-deficient mice, and in the liver and spleen of Tlr7/Tlr9-/- mice. The absence of TLR2 or TLR13 did not affect lytic viral titers. We then compared reactivation of MHV68 from latently infected WT, Unc93b-/-, Tlr7/Tlr9-/-, Tlr7-/-, and Tlr9-/- splenocytes. We observed enhanced reactivation and latent viral loads, particularly from Tlr7/Tlr9-/- splenocytes, compared to WT. Our data show that UNC93B- dependent TLR7 and TLR9 cooperate in and contribute to detection and control of MHV68 infection.
  • Regulation of MRTF-A by JMY via a nucleation-independent mechanism.

    Kluge, Franziska; Weissbach, Julia; Weber, Anja; Stradal, Theresia; Posern, Guido; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer-Nature, 2018-11-21)
    MRTF-A (myocardin-related transcription factor A) is a coactivator for SRF-mediated gene expression. The activity of MRTF-A is critically dependent on the dissociation of G-actin from N-terminal RPEL motifs. MRTF-SRF induction often correlates with enhanced polymerization of F-actin. Here we investigate MRTF regulation by the multifunctional JMY protein, which contains three WASP/verprolin homology 2 (WH2/V) domains and facilitates Arp2/3-dependent and -independent actin nucleation. Co-immunoprecipitation experiments, immunofluorescence and luciferase reporter assays were combined with selective inhibitors to investigate the effect of JMY and its domains on MRTF-A in NIH 3 T3 mouse fibroblasts. JMY induced MRTF-A transcriptional activity and enhanced its nuclear translocation. Unexpectedly, MRTF-A was hyperactivated when the Arp2/3-recruiting CA region of JMY was deleted or mutated, suggesting an autoinhibitory mechanism for full-length JMY. Moreover, isolated WH2/V domains which are unable to nucleate actin were sufficient for nuclear accumulation and SRF activation. Recombinant WH2/V regions of JMY biochemically competed with MRTF-A for actin binding. Activation of MRTF-A by JMY was unaffected by Arp3 knockdown, by an Arp2/3 inhibitor, and by latrunculin which disassembles cellular F-actin. Restriction of JMY to the nucleus abrogated its MRTF-A activation. Finally, JMY RNAi reduced basal and stimulated transcriptional activation via MRTF-A. Our results suggest that JMY activates MRTF-SRF independently of F-actin via WH2/V-mediated competition with the RPEL region for G-actin binding in the cytoplasm. Furthermore, the C-terminal region facilitates an autoinhibitory effect on full-length JMY, possibly by intramolecular folding.
  • Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non-redundant Functions in Vivo.

    Schaks, Matthias; Singh, Shashi P; Kage, Frieda; Thomason, Peter; Klünemann, Thomas; Steffen, Anika; Blankenfeldt, Wulf; Stradal, Theresia E; Insall, Robert H; Rottner, Klemens; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2018-10-25)
    Cell migration often involves the formation of sheet-like lamellipodia generated by branched actin filaments. The branches are initiated when Arp2/3 complex [1] is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family [2]. Recent structural studies defined two independent Rac binding sites on WRC within the Sra-1/PIR121 subunit of the pentameric WRC [3, 4], but the functions of these sites in vivo have remained unknown. Here we dissect the mechanism of WRC activation and the in vivo relevance of distinct Rac binding sites on Sra-1, using CRISPR/Cas9-mediated gene disruption of Sra-1 and its paralog PIR121 in murine B16-F1 cells combined with Sra-1 mutant rescue. We show that the A site, positioned adjacent to the binding region of WAVE-WCA mediating actin and Arp2/3 complex binding, is the main site for allosteric activation of WRC. In contrast, the D site toward the C terminus is dispensable for WRC activation but required for optimal lamellipodium morphology and function. These results were confirmed in evolutionarily distant Dictyostelium cells. Moreover, the phenotype seen in D site mutants was recapitulated in Rac1 E31 and F37 mutants; we conclude these residues are important for Rac-D site interaction. Finally, constitutively activated WRC was able to induce lamellipodia even after both Rac interaction sites were lost, showing that Rac interaction is not essential for membrane recruitment. Our data establish that physical interaction with Rac is required for WRC activation, in particular through the A site, but is not mandatory for WRC accumulation in the lamellipodium.
  • RhoA, Rac1, and Cdc42 differentially regulate αSMA and collagen I expression in mesenchymal stem cells.

    Ge, Jianfeng; Burnier, Laurent; Adamopoulou, Maria; Kwa, Mei Qi; Schaks, Matthias; Rottner, Klemens; Brakebusch, Cord (2018-06-15)
    Mesenchymal stem cells (MSC) are suggested to be important progenitors of myofibroblasts in fibrosis. To understand the role of Rho GTPase signaling in TGF -induced myofibroblast differentiation of MSC, we generated a novel MSC line and its descendants lacking functional Rho GTPases and Rho GTPase signaling components. Unexpectedly, our data revealed that Rho GTPase signaling is required for TGF -induced expression of -smooth muscle actin (SMA) but not of collagen I 1 (col1a1). Whereas loss of RhoA and Cdc42 reduced SMA expression, ablation of the Rac1 gene had the opposite effect. Although actin polymerization and MRTFa were crucial for TGF -induced SMA expression, neither Arp2/3-dependent actinpolymerizationnorcofilin-dependent severinganddepolymerization of F-actin were required. Instead, F-actin levels were dependent on cell contraction, and TGF -induced actin polymerization correlated with increased cell contraction mediated by RhoA and Cdc42. Finally, we observed impaired collagen I secretion in MSC lacking RhoA or Cdc42. These data give novel molecular insights into the role of Rho GTPases in TGF signaling and have implications for our understanding of MSC function in fibrosis.
  • Assembling actin filaments for protrusion.

    Rottner, Klemens; Schaks, Matthias; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2018-09-29)
    Cell migration entails a plethora of activities combining the productive exertion of protrusive and contractile forces to allow cells to push and squeeze themselves through cell clumps, interstitial tissues or tissue borders. All these activities require the generation and turnover of actin filaments that arrange into specific, subcellular structures. The most prominent structures mediating the protrusion at the leading edges of cells include lamellipodia and filopodia as well as plasma membrane blebs. Moreover, in cells migrating on planar substratum, mechanical support is being provided by an additional, more proximally located structure termed the lamella. Here, we systematically dissect the literature concerning the mechanisms driving actin filament nucleation and elongation in the best-studied protrusive structure, the lamellipodium. Recent work has shed light on open questions in lamellipodium protrusion, including the relative contributions of nucleation versus elongation to the assembly of both individual filaments and the lamellipodial network as a whole. However, much remains to be learned concerning the specificity and relevance of individual factors, their cooperation and their site-specific functions relative to the importance of global actin monomer and filament homeostasis.
  • Functional expression of TLR5 of different vertebrate species and diversification in intestinal pathogen recognition.

    Faber, Eugenia; Tedin, Karsten; Speidel, Yvonne; Brinkmann, Melanie M; Josenhans, Christine; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-07-26)
    Toll-like receptor 5 (TLR5) is activated by bacterial flagellins and plays a crucial role in the first-line defence against pathogenic bacteria and in immune homeostasis, and is highly conserved in vertebrate species. However, little comparative information is available on TLR5 functionality. In this study, we compared TLR5 activation using full-length and chimeric TLR5 of various vertebrate species (human, chicken, mouse, pig, cattle). Chimeric TLR5 receptors, consisting of human transmembrane and intracellular domains, linked to extracellular domains of animal origin, were generated and expressed. The comparison of chimeric TLR5s and their full-length counterparts revealed significant functional disparities. While porcine and chicken full-length TLR5s showed a strongly reduced functionality in human cells, all chimeric receptors were functional when challenged with TLR5 ligand Salmonella FliC. Using chimeric receptors as a tool allowed for the identification of ectodomain-dependent activation potential and partially host species-specific differences in response to various enteric bacterial strains and their purified flagellins. We conclude that both the extra- and intracellular determinants of TLR5 receptors are crucial for compatibility with the species expression background and hence for proper receptor functionality. TLR5 receptors with a common intracellular domain provide a useful system to investigate bacteria- and host-specific differences in receptor activation.
  • Early cell death induced by Clostridium difficile TcdB: Uptake and Rac1-glucosylation kinetics are decisive for cell fate.

    Beer, Lara-Antonia; Tatge, Helma; Reich, Nicole; Tenspolde, Michel; Olling, Alexandra; Goy, Sebastian; Rottner, Klemens; Alekov, Alexi Kirilov; Gerhard, Ralf; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-06-14)
    Toxin A and Toxin B (TcdA/TcdB) are large glucosyltransferases produced by Clostridium difficile. TcdB but not TcdA induces reactive oxygen species-mediated early cell death (ECD) when applied at high concentrations. We found that nonglucosylated Rac1 is essential for induction of ECD since inhibition of Rac1 impedes this effect. ECD only occurs when TcdB is rapidly endocytosed. This was shown by generation of chimeras using the trunk of TcdB from a hypervirulent strain. TcdB from hypervirulent strain has been described to translocate from endosomes at higher pH values and thus, meaning faster than reference type TcdB. Accordingly, intracellular delivery of the glucosyltransferase domain of reference TcdB by the trunk of TcdB from hypervirulent strain increased ECD. Furthermore, proton transporters such as sodium/proton exchanger (NHE) or the ClC-5 anion/proton exchanger, both of which contribute to endosomal acidification, also affected cytotoxic potency of TcdB: Specific inhibition of NHE reduced cytotoxicity, whereas transfection of cells with the endosomal anion/proton exchanger ClC-5 increased cytotoxicity of TcdB. Our data suggest that both the uptake rate of TcdB into the cytosol and the status of nonglucosylated Rac1 are key determinants that are decisive for whether ECD or delayed apoptosis is triggered.
  • Actin dynamics in host-pathogen interaction.

    Stradal, Theresia E B; Schelhaas, Mario; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-06-23)
    The actin cytoskeleton and Rho GTPase signaling to actin assembly are prime targets of bacterial and viral pathogens, simply because actin is involved in all motile and membrane remodeling processes, such as phagocytosis, macropinocytosis, endocytosis, exocytosis, vesicular trafficking and membrane fusion events, motility, and last but not least, autophagy. This article aims at providing an overview of the most prominent pathogen-induced or -hijacked actin structures, and an outlook on how future research might uncover additional, equally sophisticated interactions.
  • 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.
  • 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.
  • 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.
  • 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.

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