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Diaphanous-Related Formins: Characterization of Mouse Mdia1, and Fora/Ddia3 from Dictyostelium Discoideum Amoebae

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Diaphanous-Related Formins: Characterization of Mouse Mdia1, and Fora/Ddia3 from Dictyostelium Discoideum Amoebae Diaphanous-related formins: characterization of mouse mDia1, and ForA/dDia3 from Dictyostelium discoideum amoebae Dissertation der Fakultät für Biologie der Ludwig-Maximilians-Universität München vorgelegt von Nagendran Ramalingam München, August 2009 Ehrenwörtliche Versicherung Diese Dissertation wurde selbständig und ohne unerlaubte Hilfsmittel angefertigt. München, August 2009 Nagendran Ramalingam Erstgutachter: Prof. Dr. Michael Schleicher Zweitgutachter: Prof. Dr. Dirk Eick Eingereicht am: 12.08.2009 Tag der mündlichen Prüfung: 29.09.2009 Meeting abstracts: Ramalingam, N., Breitsprecher, D., Faix, J. and Schleicher, M. (2008) Differential regulation of mDia formins by phospholipids. The American Society for Cell Biology, San Francisco, USA, Mol. Biol. Cell, 19 (suppl), S-L94. Ramalingam, N., Rohlfs, M., Faix, J. and Schleicher, M. (2007) ForA regulates the integrity of the cortical actin cytoskeleton. The American Society for Cell Biology, Washington DC, USA, Mol. Biol. Cell, 18 (suppl), 418, B353. Ramalingam, N., Rohlfs, M., Faix, J. and Schleicher, M. (2007) ForA is a Diaphanous-related formin required for directed cell motility in Dictyostelium discoideum. The American Society for Cell Biology, Dijon, France, Dynamic interplay between cytoskeletal and membrane systems, Abstract: 17. Ramalingam, N., Faix, J. and Schleicher, M. (2006) ForA is a Diaphanous-related formin required for phototaxis and normal development of Dictyostelium cells. The American Society for Cell Biology, San Diego, USA, Mol. Biol. Cell, 17 (suppl), 839, B102. Ramalingam, N., Schleicher, M. and Faix, J. (2005) dDia3 is a membrane-associated Diaphanous-related formin involved in growth of Dictyostelium discoideum cells. German Society for Cell Biology, Heidelberg, Germany, Eur. J. Cell Biol, 84S1 (suppl. 55), S4-7. Publications: Joseph, J.M., Fey, P., Ramalingam, N., Liu, X.I., Rohlfs, M., Noegel, A.A., Mueller- Taubenberger, A., Gloeckner, G. and Schleicher, M. (2008) The actinome of Dictyostelium discoideum in comparison to actins and actin-related protein from other organisms. PLoS One, 3, e2654. Arasada, R., Son, H., Ramalingam, N., Eichinger, L., Schleicher, M., Rohlfs, M. (2006) Characterization of the Ste20-like kinase Krs1 of Dictyostelium discoideum. Eur J Cell Biol, 85, 1059-68. The experimental parts of the work presented here were carried out in the laboratory of Prof. Dr. Michael Schleicher from November 2003 to May 2009 at the Institute for Cell Biology, Ludwig-Maximilians-University, Munich. Table of contents Table of contents Summary...................................................................................................................................... 1 Zusammenfassung........................................................................................................................ 3 1 Introduction.......................................................................................................................... 5 1.1 The actin cytoskeleton................................................................................................. 5 1.2 Actin-binding proteins................................................................................................. 7 1.3 Formins ........................................................................................................................ 9 1.4 Dictyostelium discoideum as a model system............................................................ 11 1.5 Goals of the thesis...................................................................................................... 13 2 Materials and Methods....................................................................................................... 14 2.1 Materials .................................................................................................................... 14 2.1.1 Computer programs ........................................................................................... 14 2.1.2 Instruments......................................................................................................... 15 2.1.3 Reagents............................................................................................................. 15 2.1.4 Media ................................................................................................................. 17 2.1.5 Bacterial strains and D. discoideum cell lines ................................................... 17 2.1.6 Parental and recombinant plasmids ................................................................... 18 2.2 Methods...................................................................................................................... 21 2.2.1 Molecular biology.............................................................................................. 21 2.2.2 Biochemistry...................................................................................................... 21 2.2.3 Cell biology methods ......................................................................................... 26 2.2.4 Yeast-two hybrid assay...................................................................................... 27 3 Results................................................................................................................................ 30 3.1 Characterization of the mDia1-phospholipid interaction........................................... 30 3.1.1 mDia1 is a Diaphanous-related formin (DRF) from Mus musculus.................. 30 3.1.2 Domain structure of mDia1 ............................................................................... 30 3.1.3 BR is the N-terminal phospholipid-binding region in mDia1 ........................... 30 3.1.4 BR alone is sufficient for phospholipid interaction ........................................... 32 3.1.5 The first two poly-basic clusters are important for lipid-binding...................... 33 3.1.6 The N-terminal BR interacts specifically with PS............................................. 33 3.1.7 mDia1 N-terminus clusters PS and PIP2 ............................................................ 35 I Table of contents 3.1.8 mDia1 N-terminus inserts into the plasma membrane....................................... 36 3.1.9 BR is essential for plasma membrane targeting of mDia1 ................................ 37 3.1.10 PIP2-BR interaction does not influence the activity of mDia1 .......................... 40 3.1.11 PIP2 negatively regulates the activity of mDia1 ................................................ 41 3.1.12 Potential phospholipid-binding sites at the C-termini of formins...................... 46 3.1.13 DAD relieves the PIP2-mediated inhibition of mDia1 activity.......................... 46 3.1.14 mDia1FH1FH2DAD is inhibited also by unilamellar PIP2 vesicles ................. 47 3.1.15 The C-terminal region of mDia1 harbors at least two PIP2-binding sites.......... 49 3.1.16 mDia1FH1FH2DAD clusters PIP2 and inserts into the plasma membrane....... 51 3.2 Characterization of ForA ........................................................................................... 53 3.2.1 Formins from D. discoideum ............................................................................. 53 3.2.2 Domain architecture of ForA............................................................................. 55 3.2.3 ForA interaction with actin ................................................................................ 56 3.2.4 ForA interacts with profilin I ............................................................................. 57 3.2.5 Generation of a ForA null mutant...................................................................... 58 3.2.6 ForA null mutant development is largely unaffected ........................................ 59 3.2.7 ForA is essential for directed cell migration of slugs during phototaxis. .......... 60 3.2.8 ForA null mutant cells show reduced random motility ..................................... 63 3.2.9 ForA null mutant cells are proned to membrane blebbing ................................ 63 3.2.10 ForA induced blebbing is myosin II dependent................................................. 66 3.2.11 Subcellular localization of GFP-ForA and GFP-ForAΔC2 ............................... 68 3.2.12 Subcellular localization of constitutively active ForA....................................... 68 3.2.13 The C2 domain is also not required for localizing constitutively active ForA.. 71 3.2.14 GBD-FH3 harbors the localization signal.......................................................... 72 3.2.15 GFP-ForA N-terminus overexpression leads to an altered phenotype .............. 74 3.2.16 ForA localization is myosin II independent....................................................... 74 3.2.17 Putative binding partners of ForA...................................................................... 75 3.3 Characterization of dDia3 .......................................................................................... 77 3.3.1 dDia3 is a DRF from Dictyostelium discoideum ............................................... 77 3.3.2 Recombinant dDia3 nucleates and caps actin filaments .................................... 79 3.3.3 dDia3 interacts with all three profilin isoforms ................................................. 79 II Table of contents 3.3.4 Characterization of dDia3 null mutant cells ...................................................... 80 3.3.5 Subcellular localization of GFP-dDia3.............................................................
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