3D-structure of actin: cofilin and nuclear translocation of actin and thymosin ß4
Zusammenfassung der Projektergebnisse
The protein actin is an essential component of the cytoskeleton and as such involved in cellular activities like cell migration, intracellular transport of vesicles, cell division, and exoand endocytosis. According to the cellular activities the intracellular actin cytoskeleton is reorganized, i.e. new actin-filaments are formed or preexisting filaments are disassembled. These dynamic reorganisational processes are initiated and performed by a large number of actin binding proteins (AbP). Necessary for the formation of new filaments is a sufficiently large pool of unpolymerized actin. The AbPs mainly responsible to the maintainance of the intracellular pool of monomeric actin is thymosin ß4 (Tß4). Proteins of the ADF (actin depolymerizing factor) and cofilin can under certains conditions promote the disassembly of F-actin filaments. Monoclonal antibodies and a number of vectors coding for differently tagged Tß4 were used to analyse the intracellular distribution of Tß4. The data demonstrated a peripheral and a cell line dependent nuclear localisation of Tß4. Using purified proteins a cooperative behaviour of Tß4 with ADF/cofilin was shown for F-actin disassembly. Photoreactive derivatives of Tß4 were synthetized with the aim to identify additional binding partners of Tß4 in cytoplasmic und nuclear extracts. The importance of the Tß4 maintained intracellular equilibrium betqween monomeric and filamentous actin was shown by the effect of the TccC3-toxin of the entomopathogenic bacterium Photorhabdus luminescens, which ADP-ribosylates actin at threonine 148. Photorhabdus luminescens lives symbiotically in nematodes and infects insect larvae, but infections of humans have been reported in the USA and Australia. Threonine 148 forms part of the Tß4 binding area of actin. Biochemical and stopped flow experiments indicated a reduced affinity of this modified actin for Tß4 to such an extent that its diminished ability to interact with Tß4 results in the formation of stable intracellular actin bundles and finally cell death. These data underlined the importance of a finely balanced equilibrium between monomeric actin stabilizing and polymerization promoting AbPs. In addition, the effect of point mutations of cardiac actin on its functionality was investigated. The ability to polymerize and to stimulate the cardiac mayosin ATPase activity was clearly decreased for the two analysed mutant actins. Furthermore, mixtures of wild type actin with a mutant actin led to an impairment of these activities suggesting a mechanism for the action of these point mutations cardiac actin identified in patients with hypertrophic cardiomyopathy.
Projektbezogene Publikationen (Auswahl)
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(2009): Gelsolin in human colon adenocarcinoma cells with different metastatic potential. Acta Biochim Pol. 56, 739-743
Litwin, M., Mazur, A.J., Nowak, D., Mannherz, H.G., Malicka-Błaszkiewicz, M.
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(2009): The ß-thymosins: Intracellular and extracellular activities of a versatile actin binding protein family. Cell Motil. Cytoskeleton 66, 839-851
Mannherz, H.G. and Hannappel, E.
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(2010): Actin filament bundling and different nucleating effects on actin:ADF and actin:cofilin complexes by FH2-domains of mouse Diaphanous related formins. J. Mol. Biol. 403; 529-545
Machaidze, G., Sokoll, A., Shimada, A., Lustig, A., Mazur, A., Wittinghofer, A., Aebi, U., and Mannherz, H.G.
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(2010): Photorhabdus luminescens Toxins ADP-Ribosylate Actin and RhoA to Force Actin Clustering. Science 327, 1139-1142
Lang, A.E., Schmidt, G., Schlosser, A., Hey, T.D., Larrinua, I.M., Sheets, J.J., Mannherz, H.G., Aktories, K.
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(2011): Actin as target for modification by bacterial protein toxins. FEBS J. 278, 1-18
Aktories, K., Lang, A.E., Schwan, C., and Mannherz, H.G.
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(2011): Actin: From structural plasticity to functional diversity. Eur. J. Cell Biol. 90, 797-804
Schoenenberger, C.A., Mannherz, H.G., and Jockusch, B.M.
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(2012): Functional characterization of the human α-cardiac actin mutations Y166C and M305L involved in hypertrophic cardiomyopathy. Cellul. Molec. Life Sci. 69; 3457-3479
Müller, M., Mazur, J.A., Behrmann, E., Diensthuber, R.P., Radke, M. P., Qu, Z., Littwitz, C., Raunser, S., Schoenenberger, C.-A., Manstein, D.J., and Mannherz, H.G.
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(2012): Identification of interaction partners of β-thymosins: application of thymosin β4 labeled by transglutaminase. Ann N Y Acad Sci.1270; 98-104
App, C., Knop, J., Mannherz, H.G., and Hannappel, E.
