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Projects

The central theme of my laboratory’s research consist of virus-host interactions with a strong focus on the cell biology of motile events such as viral egress, intracellular trafficking and cell motility. These interests are pursued in three main areas of research that integrate state of the art experimental approaches in cell biology, molecular biology and virology. The primary focus of our studies historically consists of the molecular mechanisms of action of the HIV-1 pathogenicity factor Nef. More recently we have started to address how the intrinsic immunity factor CD317/BST-2/thetherin impairs HIV-1 particle release and how this activity is antagonized by the viral protein Vpu. Finally, studies on the modulation of the host cell cytoskeleton architecture by Nef triggered our interest in mechanisms by which mammalian cells control and modulate the organization of their actin cytoskeleton. This resulted in the generation of a research goal consisting in the characterization of the molecular biology and physiological role of the actin-nucleating diaphanous-related formins (DRFs). In this context we particularly focus on the formation of non-apoptotic plasma membrane blebs that are instrumental for certain types of motility and invasion modes of tumour cells.

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1) Molecular mechanisms of the HIV-1 pathogenicity factor Nef

The Nef protein of HIV and SIV is critical for high virus load and full pathogenicity in the infected host. This pathogenic potential is underscored by a transgenic mouse model in which the expression of Nef alone causes an AIDS-like disease. This activity renders Nef an attractive target for antiviral strategies to complement existing treatment. Nef does not posses detectable enzymatic activity and mediates all its functions via interactions with host cell or other viral proteins. While a multitude of activities have been ascribed to Nef, the biological properties and protein-interactions that govern its pathogenic potential have still not been unambiguously identified. Nef`s activities can be divided into immune evasion functions and effects that directly boost viral spread in the absence of host immune attacks. Both these general Nef activities are achieved by manipulation of host cell signal transduction and intracellular transport processes. For example, Nef interferes with CTL recognition and antigen presentation by regulating cell surface presentation of MHC-I and MHC-II complexes in infected cells. Nef also prevents potentially detrimental consequences for productively infected cells by downmodulating the HIV entry receptor-coreceptor complex. Finally, Nef alters intracellular signaling pathways to sensitize infected cells for activation, presumably in order to induce an intermediate state of activation that is permissive to HIV replication. The current view in the field predicts that the pathogenic potential results from the integration of multiple of its effector functions in the infected host. Which of its biological properties are pathologically relevant and which exact molecular mechanisms Nef employs to manipulate host cells still remains largely unclear. In T lymphocytes, one pathway that is particularly affected by Nef is T cell receptor (TCR) signal transduction. We and others have studied over the past years the interactions of T lymphocytes in close contact with antigen presenting cells, the immunological synapse (IS), at which TCR signaling is initiated and regulated. Our recent studies revealed that Nef potently affects protein sorting (e.g. intracellular accumulation of the kinase Lck; Fig. 1) and actin remodeling at the immunological synapse (Fig. 2), presumably resulting in a reduced TCR signal that renders target T lymphocytes permissive for HIV replication but prevents premature activation induced cell death. The precise mechanisms of IS modulation by Nef as well as their individual functional consequences are the focus of our current work in this area.

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Fig. 1: Lck kinase is translocated into an intracellular membrane compartment in the presence of Nef.GFP

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Fig. 2: Raji B cells that were pulsed by SEE superantigen (in blue) form close contacts (IS) with T lymphocytes infected with HIV-1Dnef (in green, left hand picture), allowing for polymerization of F-actin at the cell-cell contact. In the presence of Nef (HIV-1 wt, right hand picture), IS formation is disturbed and no F-actin polymerization at the cell-cell contact can be observed.

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2) The intrinsic immunity factor CD317 and antagonism by HIV-1 Vpu

Mammals have evolved a set of cellular defense mechanisms that, often upon induction by type-I interferons (INF), are capable of inhibiting the replication of viral pathogens. These restriction factors impose particularly effective barriers in the context of cross-species transmission of viruses and are thought to act as “intrinsic” immunity defense in an immunologically naïve host. As an important characteristic of such factors, fine-tuned evolution gave raise to highly species-specific adaptation between host and pathogen. Two recent landmark studies by the Bieniasz and Guatelli laboratories identified CD317 (also referred to as BST-2/HM1.24/tetherin) as a novel host cell innate immunity factor that potently blocks the release of mature HIV-1 particles but also that of other retroviruses as well as Ebola, Marburg, and Lassa virus from productively infected cells. CD317 is an INF-α-inducible, glycosylated transmembrane protein with an unusual topology consisting of a short cytoplasmic tail, a transmembrane domain and a large extracellular domain that is attached to membranes via its GPI anchor. Localized at the plasma membrane as well as in yet to be defined endosomal compartments, CD317 at least partially resides in detergent resistant membrane microdomains. Expression of CD317 potently prevents the final release of fully matured HIV particles from infected cells through surface-protein-based tethers (Fig. 4). To circumvent this antiviral defense, viral pathogens acquired antagonists that counteract the replication block imposed by CD317. These include the proteins Vpu and Env for HIV-1 and -2 as well as the K5 and envelope glycoproteins of KSHV and Ebola viruses. The molecular bases for CD317 mediated restriction, viral antagonism and the species specificity of these processes remains to be determined and are subject of our current studies.

 

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Fig. 4: Subcellular localization of p24CA and CD317/tetherin in human T lymphocytes infected with HIV-1. Infection with HIV-1Dvpu results in characteristic accumulation (tethering) of p24CA at the cell surface. Expression of Vpu from HIV-1 wt causes a reduction of CD317 expression and allows for “normal” p24Gag distribution and HIV-1 particle release.

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The astonishing dynamics of the actin cytoskeleton provides the basis for fundamental processes in mammalian cells such as motility, kinesis, vesicular transport and signal transduction. This includes the regulation of non-apoptotic plasma membrane (PM) blebs, cellular protrusions often observed during stem cell motility, cytokinesis and cancer cell invasion. They are highly dynamic structures that depend on the regulation of the actin cytoskeleton, which controls bleb expansion, stabilization and retraction. PM blebbing is initiated by local disruption of the membrane-actin cortex leading to rapid protrusion of the PM due to cytosolic streaming and subsequent local increase in cortical contractility. While the expanding bleb seems initially not coupled to an actin cortex, actin is subsequently polymerized at the bleb cortex to halt bleb expansion. Then, acto-myosin contractility can be generated to retract the bleb. Dynamic PM blebbing therefore critically and essentially depends on F-actin assembly and integrity (Fig. 5).

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We are currently investigating the identity and functional role of individual actin nucleators in various cellular models of PM blebbing in our to dissect the molecular regulation of blebbing and to define the functional role of PM blebbing.

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Fig. 5: Schematic model of dynamic plasma membrane blebbing

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Selected Publications

Complete Publication List (PubMed)

 

Fackler, O.T. *, Murooka, T.T., Imle, A. and Mempel, T.R.* Adding new dimensions: Towards an integrative understanding of HIV-1 spread. (* corresponding authors). Nat. Rev. Microbiol., in press.

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Kutscheidt, S+., Zhu, R. +, Antoku, S. +, Luxton, G.G.W., Stagljar, I., Fackler, O.T. * and Gundersen, G. * (2014). FHOD1 interaction with nesprin-2G mediates TAN line formation and nuclear movement (+ first authors, * corresponding authors). Nat. Cell Biol. 16: 708-715.

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Geist, M.M., Pan, X., Bender, S., Bartenschlager, R., Nickel, W and Fackler, O.T. (2014). Heterologous Src Homology 4 Domains Support Membrane Anchoring and Biological Activity of HIV-1 Nef. J. Biol. Chem., Apr 4. [Epub ahead of print] PMID: 24706755

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Fackler, O.T. and Keppler, O.T. (2013). MxB/Mx2: The latest piece in HIV’s interferon puzzle. Embo Rep. 14: 1030 – 1031.

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Pan, X., Baldauf, H.M., Keppler, O.T. and Fackler, O.T. (2013). Restrictions to HIV-1 Replication in Resting CD4+ T Lymphocytes. Cell Research, 23: 876-885.

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Floyd, S., Whiffin, N., Gavilan, M.P., Kutscheidt, S., De Luca, M., Watkins, J., Chung, K., Fackler, O.T. and Lindon, C. (2013). Spatiotemporal organization of Aurora-B by APC/C-Cdh1 coordinates cell spreading via FHOD1 after mitosis. J. Cell Sci., 126: 2845-2856.

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Schönichen, A., Mannherz, H.G., Behrmann, E., Mazur, A.J., Kühn, S., Silvan, U., Schoenenberger, C.A., Fackler, O.T., Raunser, S., Dehmelt, L. and Geyer, M. (2013). FHOD1 is a combined actin filament capping and bundling factor that selectively associates with actin arcs and stress fibers. J. Cell Sci. 126: 1891-1901.

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Pan, X., Geist, M., Rudolph, J.M., Nickel, W. and Fackler, O.T. (2013). HIV-1 Nef Disrupts Membrane-Microdomain-associated Anterograde Transport for Plasma Membrane Delivery of Selected Src Family Kinases. Cell. Microbiol., 15:1605-1621.

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Baldauf, H-M.+, Pan, X.+, Erikson, E., Schmidt, S., Daddacha, W., Burggraf, M., Schenkova, K., Ambiel, I., Wabnitz G., Gramberg, T., Panitz, S., Flory, E., Landau, N.R., Sertel, S., Rutsch, F., Lasitschka, F., Kim, B., König, R., Fackler, O.T.* and Keppler, O.T.* (2012). The deoxynucleoside triphosphate triphosphohydrolase SAMHD1 restricts HIV-1 infection in resting CD4+ T cells. Nat. Med., 18: 1682-1687 (* corresponding authors, + first authors).

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Stolp, B., Imle, A., Coelho, F.M., Hons, M., Mendiz, R.G., Lyck, R., Stein, J.V. and Fackler, O.T. (2012). HIV-1 Nef Interferes With T Lymphocyte Circulation Through Confined Environments in vivo. Proc. Natl. Acad. Sci. USA, 109: 18541–18546.

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Abraham, L. and Fackler, O.T. (2012). HIV-1 Nef: a multifaceted modulator of TCR signaling. Cell Communication and Signaling, 10:39.

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Sauter, D., Unterweger, D., Vogl, M., Usmani, S.M., Heigele, A., Kluge, S.F., Hermkes, E., Moll, M., Barker, E., Peeters, M., Learn, G.H., Fritz, J.V., Fackler, O.T., Hahn, B.H. and Kirchhoff, F. (2012). Human Tetherin Exerts Strong Selection Pressure on the HIV-1 Group N Vpu Protein. PLoS Pathogens, 8: e1003093.

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Götz, N., Sauter, D., Usmani, S.M., Fritz, J.V., Goffinet, C., Heigele, A., Geyer, M., Bibollet-Ruche, F., Learn, G.H., Fackler, O.T., Hahn, B.H. and Kirchhoff, F. (2012). Reacquisition of Nef-Mediated Tetherin Antagonism in a Single In Vivo Passage of HIV-1 Through Its Original Chimpanzee Host. Cell Host Microbe, 12: 373-380.

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Abraham, A., Bankhead, P., Pan, X., Engel, U. and Fackler, O.T. (2012). HIV-1 Nef Limits Communication Between LAT and SLP-76 to Reduce Formation of SLP-76 Signaling Microclusters Following TCR Stimulation. J. Immunol., 189: 1898–1910.

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Meuwissen, P. J., Stolp, B., Ianucci, V., Vermeire, J., Naessens, E., Saksela, K., Geyer, M., Vanham, G., Arien, K., Fackler, O.T. and Verhasselt, B. (2012). Identification of a highly conserved valine-glycine-phenylalanine amino acid triplet required for HIV-1 Nef function. Retrovirology, 9: 34.

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Fritz, J.V., Tibroni, N., Keppler, O.T. and Fackler, O.T. (2012). Membrane Microdomain Association of HIV-1 Vpu is Dispensable for Counteracting the Particle Release Restriction Imposed by CD317/BST-2/Tetherin. Virology, 424: 33–44.

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Pan, X., Rudolph, J.M., Abraham, L., Habermann, A., Haller,C., Krijnse-Locker, J. and Fackler, O.T. (2012). HIV-1 Nef Compensates Disorganization of the Immunological Synapse by Assembly of an Intracellular Lck Signalosome. Blood; 119: 786-797.

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Stastna, J., Pan, X.Y., Wang, H., Kollmannsperger, A., Kutscheid, S., Lohmann, V., Grosse, R. and Fackler, O.T. (2012). Differing and isoform specific roles for the formin DIAPH3 in plasma membrane blebbing and filopodia formation. Cell Research, 22: 728–745.

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Schmidt, S., Fritz, J.V., Bitzegeio, J., Fackler, O.T. *, and Keppler, O.T*. (2011). HIV-1 Vpu Blocks Recycling and Biosynthetic Transport of the Intrinsic Immunity Factor CD317/Tetherin to Overcome the Virion Release Restriction. (* corresponding authors). mBio, 2: e00036-11.

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Breuer, S., Schievink, S.I., Schulte, A., Blankenfeldt, W., Fackler, O.T. * and Geyer, M.* (2011). Molecular design, functional characterization, and structural basis of a protein inhibitor against the HIV-1 pathogenicity factor Nef. (* corresponding authors). PlosOne, 6: e20033.

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Stolp, B. and Fackler, O.T. (2011). How HIV takes advantage of the cytoskeleton in entry and replication. Viruses, 3: 293-311.

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Bouchet, J., Basmaciogullari, S., Chrobak, P., Stolp, B., Bouchard, N., Fackler, O.T., Chames, P., Jolicoeur, P., Benichou, S. and Baty, D. (2011). Inhibition of the Nef Regulatory Protein of HIV-1 by a Single-Domain Antibody. Blood, 117: 3559-3568.

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Tervo, H.M., Homann, S., Ambiel, I., Fritz, J.V., Fackler, O.T. * and Keppler, O.T. * (2011). ß-TrCP is Dispensable for Vpu’s Ability to overcome the CD317/Tetherin-imposed restriction to HIV-1 release. (* corresponding authors). Retrovirology, 8:9.

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Goffinet, C., Homann, S., Ambiel, I., Tibroni, N., Rupp, D., Keppler O.T. and Fackler, O.T. (2010). Antagonism of CD317 restriction of HIV-1 particle release and depletion of CD317 are separable activities of HIV-1 Vpu. J. Virol., 84: 4089-4094.

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Stolp, B., Abraham, L., Rudolph, J.M. and Fackler, O.T. (2010). Lentiviral Nef Proteins Utilize PAK2-mediated Deregulation of Cofilin as a General Strategy to Interfere with Actin Remodeling. J. Virol., 84: 3935-3948.

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Stolp, B., Abraham, L., Rudolph, J.M. and Fackler, O.T. (2010). Lentiviral Nef Proteins Utilize PAK2-mediated Deregulation of Cofilin as a General Strategy to Interfere with Actin Remodeling. J. Virol., 84: 3935-3948.

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Rudolph, J.M.; Eickel, N., Haller, C., Schindler, M. and Fackler, O.T. (2009). Inhibition of T cell receptor induced actin remodeling and relocalization of Lck are evolutionarily conserved activities of lentiviral Nef proteins. J. Virol., 83: 11528-11539.

Stolp, B., Raichman-Fried, M., Abraham. L., Pan, X., Giese, S.I., Hannemann, S., Goulimari, P., Raz, E., Grosse, R. and Fackler, O.T. (2009). HIV-1 Nef interferes with host cell motility by deregulation of cofilin. Cell Host and Microbe 6:174-186

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Goffinet, C., Allespach, A, Homann, S., Tervo, H-M., Habermann, A., Rupp, D., Oberbremer, L., Kern, C., Tibroni, N., Welsch, S., Krijnse-Locker, J., Banting, G., Kräusslich, H.G., Fackler, O.T. and Keppler, O.T. (2009). HIV-1 Antagonism of CD317 is Species-Specific and Involves Vpu-Mediated Proteasomal Degradation of the Restriction Factor. Cell Host and Microbe, 5: 285-297.

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Homann, S., Tibroni, N., Baumann, I., Sertel, S., Keppler, O.T. and Fackler, O.T. (2009). Determinants in HIV-1 Nef for enhancement of viral replication and depletion of CD4+ T-lymphocytes in human lymphoid tissue ex vivo. Retrovirology, 6: 6.

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Schulte, A., Stolp, B., Schönichen, A., Pylypenko, O., Rak, A., Fackler, O.T. and Geyer, M. (2008). Structure of the N-terminal region of the formin FHOD1 reveals a bipartite organization of FH3 and GTPase-binding domains. Structure, 16:1313-1323.

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Hannemann, S., Madrid, R., Stastna, J., Kitzing, T., Gasteier, J., Schönichen, A., Bouchet, J., Jimenez, A., Geyer, M., Grosse, R., Benichou, S. and Fackler, O.T. The Diaphanous Related Formin FHOD1 Associates with ROCK1 and Promotes Src-dependent Plasma Membrane Blebbing. J. Biol. Chem., 283: 27891-27903.

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Haller, C. and Fackler, O.T. (2008). HIV-1 at the immunological and T lymphocytic virological synapse. Biol. Chem., 389: 1253-1260

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Fackler, O.T. and Grosse, R. (2008). Cell motility through plasma membrane blebbing. J. Cell Biol. 181: 879 

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Rauch, S., Pulkkinen, K., Saksela, K., and Fackler, O.T. (2008). HIV-1 Nef Recruits the Guanine Exchange Factor Vav1 via an Unexpected Interface into Plasma Membrane Microdomains for Association with Pak2 Activity. J. Virol., 82: 2918-2929.

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Haller, C., Rauch, S. and Fackler, O.T. (2007). HIV-1 Nef employs two distinct mechanisms to modulate Lck subcellular localization and TCR induced actin remodeling. PLoSONE, 2: e1212

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Tournaviti, S., Hannemann, S., Terjung, S., Kitzing, T.M., Stegmayer, C., Ritzerfeld, J., Walther, P., Grosse, R., Nickel, W. and Fackler, O.T. (2007). SH4 Domain-induced Plasma Membrane Dynamization Promotes bleb-associated Cell Motility. J. Cell Sci. 120: 3820-3829.

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Brügger, B., Krautkrämer, E., Tibroni, N., Munte, C.E., Rauch, S., Leibrecht, I., Glass, B., Breuer, S., Geyer, M., Kräusslich, H.G., Kalbitzer, H.R., Wieland, F.T. and Fackler, O.T. (2007). Human Immunodeficiency Virus Type 1 Nef Protein Modulates the Lipid Composition of Virions and Host Cell Membrane Microdomains. Retrovirology, 4: 70.

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Kitzing, T.M., Sahadevan, A.S., Brandt, D.T., Knieling, H., Hannemann, S., Fackler, O.T., Großhans, J. and Grosse, R. (2007). Positive feedback between Dia1, LARG and RhoA is required for cancer cell invasion. Genes and Development 21: 1478 - 1483.

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Fackler, O.T.*, Alcover, A. and Schwartz, O.* (2007). Modulation of the Immunological Synapse: A key to HIV-1 pathogenesis? (* corresponding authors). Nat. Rev. Immunol. 7: 310-317.

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Rauch, S. and Fackler, O.T. (2007). Lipid Raft as Targets for the Modulation of Host Cell Signal Transduction by Viral Pathogens. Signal Transduction 7: 53-63.

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Haller, C., Rauch, S., Michel, N., Hannemann, S., Keppler, O.T. and Fackler, O.T. (2006). The HIV-1 pathogenicity factor Nef interferes with maturation of stimulatory T-lymphocyte contacts by modulation of N-Wasp activity. J. Biol. Chem. 281: 19618-19630.

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Fackler, O.T.*, Moris, A., Tibroni, N., Giese, S.I., Glass, B., Schwartz, O. and Kräusslich, H.G. (2006). Functional characterization of HIV-1 Nef mutants in the context of viral infection. (* corresponding author). Virology 351: 332-339.

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Gasteier J.E., Madrid R., Krautkrämer E., Schröder S., Muranyi W., Benichou S. and Fackler O.T. (2003). Activation of the Rac interaction partner formin homology 2 domain containing 1 (FHOD1) induces actin stress fibers via a ROCK dependent mechanism. J. Biol. Chem. 278: 38902-38912.

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Fackler O.T. and Baur A.S. (2002). Live and let die: Functions of Nef beyond HIV replication. Immunity 16: 493-497.

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Geyer M., Fackler O.T., and Peterlin B.M. (2001). Structure-Function Relationship in HIV-1 Nef. EMBO Rep. 2: 580-585.

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Fackler O.T., Luo W., Geyer M., Alberts A.S. and Peterlin B.M. (1999). Activation of Vav by Nef induces cytoskeletal rearrangements and downstream effector functions. Mol. Cell 3: 729-739.

 

 

 

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Group members

Oliver T. Fackler, Ph.D., Prof.

Email: oliver.fackler@med.uni-heidelberg.de

Phone: ++49-(0)6221-56-1322

Fax: ++49-(0)6221-56-5003

 

 


Amanda Chase, postdoc Email: amanda.chase@med.uni-heidelberg.de Phone: ++49-(0)6221-56-1321

Johanna Galaski, MD student Email: johanna.galaski@med.uni-heidelberg.de Phone ++49-(0)6221-56-1327

Miriam Geist, PhD student Email: miriam.geist@med.uni-heidelberg.de Phone: ++49-(0)6221-56-1327 

Claudia Haller, project coordinator Email: claudia.haller@med.uni-heidelberg.de Phone: ++49-(0)6221-56-5007

Andrea Imle, PhD student Email: andrea.imle@med.uni-heidelberg.de Phone: ++49-(0)6221-56-1327 

Stefan Kutscheidt, PhD student Email: stefan.kutscheidt@med.uni-heidelberg.de Phone: ++49-(0)6221-56-1321 

Miguel Lamas, PhD student Email: miguel.lamas@med.uni-heidelberg.de Phone: ++49-(0)6221-56-1327    

Birthe Müller, PhD student Email: Birthe.Mueller@med.uni-heidelberg.de Phone: ++49-(0)6221-56-1327 

Francois Pujol, postdoc Email: Francois.Pujol@med.uni-heidelberg.de Phone: ++49-(0)6221-56-1327

Tatjana Reif, PhD student Email: tatjana.reif@med.uni-heidelberg.de Phone: ++49-(0)6221-56-1321 

Bettina Stolp, postdoc Email: bettina.stolp@med.uni-heidelberg.de Phone: ++49-(0)6221-56-1327

Nadine Tibroni, research associate Email: nadine.tibroni@med.uni-heidelberg.de Phone: ++49-(0)6221-56-1327 

Maud Trotard, postdoc Email: Maud.Trotard@med.uni-heidelberg.de Phone: ++49-(0)6221-56-1321 

Nikolaos Tsopoulidis, PhD student Email: Nikolaos.Tsopoulidis@med.uni-heidelberg.de Phone: ++49-(0)6221-56-1321 

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Former lab members: 

Libin Abraham (postdoc, University of British Colombia, Vancouver)

Jingyan Chen

Judith Gasteier (technical leader, Abbot)

Simone Giese 

Sebastian Hannemann (postdoc, Yale University)

Stefanie Homann (postdoc, University of California, San Diego)

Ellen Krautkrämer (postdoc, University Hospital Heidelberg)

Joelle Fritz (postdoc, University of Luxembourg)

Xiao-Yu Pan (senior scientist, manager, Boehringer Ingelheim, Shanghai)

Susanne Rauch (postdoc, King's College, London)

Jochen Rudolph  (postdoc, Universität Magdeburg)

Jana Stastna

Ilka Wörz 

Yaz Woo

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