Dokument

Summary:
The somatic musculature of Drosophila is analogous to vertebrate skeletal muscles and is generated by the fusion of mononucleate myoblasts. Muscle fusion in Drosophila involves two distinct cell populations, founder cells (FCs) and fusion-competent myoblasts (FCMs). The recognition and adhesion of both myoblast types is mediated by members of the Immunoglobin superfamily (IgSF) that are expressed myoblast type specifically: Duf and Rst are expressed in FCs. Sns, Hbs and Rst are present in FCMs. The heterophilic interaction of these Ig-domain proteins leads to signal activation and results in the formation of F-actin at the sites of cell-cell contact. Duf and Rst serve in functional redundancy in FCs. The formation of new actin filaments at existing filaments is regulated by the actin-related protein complex (Arp)2/3. The Arp2/3 complex is a multiprotein complex consisting of seven subunits including Arp2 and Arp3. The complex becomes activated by nucleation promoting factors (NPFs), i.e the Wiskott-Aldrich syndrome protein (WASP) and the suppressor of CAMP-receptor (SCAR). Additionally, WASP is activated by the WASP-interacting protein Verprolin1 (WIP). These proteins act together in the WASP-WIP complex. Although WASP and SCAR are activated differently to induce Arp2/3-mediated actin polymerization, they share a common proline-rich region, which is known to bind to SH3 domain containing proteins. In this study I have addressed two questions. First, I have tried to elucidate the redundant nature of Duf and Rst in FCs. The intracellular domain of Rst and Duf contains three conserved domains, i.e a PADVI, SAIYGNPYLR and NSLLPPLPP domain. Expression of RstΔPADVI in all myoblasts or exclusively in FCs as well as FCMs impaired myoblast fusion. This indicates that the PADVI domain in the intracellular domain of Rst plays an important role during myoblast fusion. To identify Rst specific interaction partners I further performed a yeast two-hybrid screen and identified Actin57B, Papilin and Nidogen as possible Rst interaction partner. Second, I investigated the role of the SH2-SH3 adopter Dreadlock (Dock) in myoblast fusion. Dock is expressed in FCs and FCMs. In vertebrates, the homologue of Dock - called Nck - links cell adhesion during podocyte formation with actin cytoskeleton rearrangement by binding to the intracellular domain of Nephrin. Nephrin is a homologue of Drosophila Sns and Hbs. Biochemical data provided in this study show that Dock is able to bind to the intracellular domain of Sns, Hbs, Duf and Rst and to the proline-rich region of the actin regulators WASP and WIP via its SH3 domains. Interestingly, I found that the SH2 domain of Dock binds to phosphorylated tyrosine at position 1089 in the intracellular domain of Hbs. The SH3 domains of Dock can bind to Sns and Duf and all SH2 and SH3 domains are required to bind to Rst. To demonstrate that these protein interactions are relevant for myoblast fusion, I carried out double mutant experiments. I could show that dock interacts genetically with the FC-specific gene rols, which encodes for an adaptor protein that binds to the intracellular domain of Duf, and with the FCM-specific cell adhesion molecule hbs. These data indicate that Dock functions in both myoblast populations during myoblast fusion and serves as a linker to transfer the fusion signal from the cell adhesion molecules Duf and Hbs to the actin cytoskeleton, e.g. by interacting with WASP and WIP.

Bibliographie / References

1. Mayer, B. J., Jackson, P. K. and Baltimore, D. (1991). The noncatalytic src homology region 2 segment of abl tyrosine kinase binds to tyrosine-phosphorylated cellular proteins with high affinity. Pro Natl Acad Sci U S A 2, 627-31.
2. Kinley, A. W., Weed, S. A., Weaver, A. M., karginov, A.V., Bissonette, E., Cooper, J.A. and Parsons J.T. (2003). Cortactin interacts with WIP in regulating Arp2/3 activation and membrane protrusion. Curr Biol. 5, 384-93.
3. Mutations in a novel gene, myoblast city, provide evidence in support of the founder cell hypothesis for Drosophila muscle development. Development. 7, 1979-88.
4. Paululat, A., Burchard, S. and Renkawitz- Pohl, R (1995). Fusion from myoblasts to myotubes is dependent on the rolling stone gene (rost) of Drosophila. Development 121, 2611-20.
5. Dworak, H. A., Charles, M. A., Pellerano, L. B and Sink, H. (2001). Characterization of Drosophila hibris, a gene related to human nephrin. Development 128, 4265-76.
6. Kesper, D. A., Stute, C., Buttgereit, D., KreiskÖther, N., Vishnu, S., Fischbach, K. F. and Renkawitz-Pohl, R. (2007). Myoblast fusion in Drosophila melanogaster is mediated through a fusion-restricted myogenic- adhesive structure (FuRMAS). Dev Dyn 236, 404-15
7. The irregular chiasm C-roughest locus of Drosophila, which affects axonal projections and programmed cell death, encodes a novel immunoglobulin-like protein. Gene Dev 7(12B): 2533-2547.
8. Luo, l., Liao, Y. J., Jan, L., and Jan, Y. N. (1994). Distinct morphogenetic function of similar GTPase: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion.
9. Giot, L., Bader, S., Brouwer, C., Chadhuri, A., Kunang, Li, Y., hao, Y.L., Ooi, C,E., Godwin, B., Vitols, E., Vijayadamodar, G., Pochart., Machineni, H.et al. and Rohthberg, J.M. (2003). A protein interaction map of Drosophila melanogaster. Science 302, 1727- 36.
10. Richardson, B.E., Beckett, Nowak, S.J. and Baylies, M. K. (2007). SCAR/WAVE and Arp2/3 are crucial for cytoskeletal remodelling at the site of myoblast fusion. Development 134, 4357-67.
11. Kim, S.,Shilagardi, K., Zhang, S., Hong, S. N., Sens, K. L., Bo, J., Gonzalez, G.A. and Chen, E. H. (2007). A critical function for the actin cytoskeleton in targeted exocytosis of prefusion vesicles during myoblast fusion. Dev Cell 12, 571-86.
12. Clemens, J., Ursuliak, Z., Clemens, K.K., Price, J. V. and Dixon, J.E. (1996). A Drosophila protein-tyrosine phosphatise associates with an adaptor protein required for axonal guidance. J Biol Chem. 29, 17002-5.
13. Cell fusion during development. Trends Cell Biol 17, 537-46.
14. Hummel, T., menne, T., Scholz, H., Granderrath, S., Giessen, K., and Klämbt, C. (1997). CNS midline map development in Drosophila. Perspect Dev Neurobiol. 4, 357-68.
15. Control of cell fates and segmentation in the Drosophila mesoderm. Development 124, 2915-2922.
16. Menon, S. D and Chia, W. (2001). Drosophila Rolling pebbles: a multidomain protein required for myoblast fusion that recur D-Titin in response to the myoblast attractant Dumbfounded. Dev Cell 1, 691-703.
17. Horsley, V. and Pavlath, G. K (2004). Forming a multinucleated cell: molecules that regulate myoblast fusion. Cells Tissues Organs 176, 67- 78
18. Kitamura, Y., Kitamura., T., Sakaue, H.,bMaeda, T., Ueno, H., Nishio, S., Osada, S., Sakaue, M Ogawa, W., Kasuga, M. (1997). Interaction of Nck -associated protein 1 with activated GTP-binding protein Rac. Biochem J. 3, 873-8.
19. Isolation of a novel gene mutated in Wiskott- Aldrich syndrome. Cell, 79 (5), 922.
20. Kitamura, T., Kitamura., Y., Yonezawa, K., Totty, N.F., Gout, I., Hara, K., Waterfield, M.D., Sakaue, M., Ogawa, W., Kasuga, M. (1996). Molecular cloning of P125Nap1, a Preotein that assaociates with an SH3 domain of Nck. Biochem Biophys Res commun. 2, 509- 14.
21. Dworak, H. A. and Sink H. (2002). Myoblast fusion in Drosophila. Bio-Essays 24, 591-601
22. Ruiz-Gomez, M., Coutts, N., Suster, M. L., Landgraf, M. and Bate, M. (2002). Myoblasts Incompetent encodes a zinc finger transcription factor required to specify fusion- competent myoblasts in Drosophila. Development 129, 133-141.
23. Myoblast vity, the Drosophila homolog of DOCK180/CED-5, is required in Rac signalling pathway utilized for multiple developmental processes. Genes and Dev 12, 3337-3342.
24. Jones, N., Blassutig, I.M., Eremina, V., Ruston, J.M., Bladt, F., Li, H., Huang, H., larose, L., Li, S.S., Takano, T., Quaggin, S.E., and Pawson, T. (2006). Nck adaptor proteins link nephrin to the actin cytoskeleton of Kidney podocytes. Nature 440, 818-23.
25. Rohatgi, R., Nollau, P., Ho, H. Y., Kirschner, M. W. And Mayer, B.J (2001). Nck and phosphatidylinositol 4,5-bisphosphate synergistically activate actin polymerization through the N-WASP-Arp2/3 pathway. J Biol Chem. 28, 26448-52.
26. Furlong, E. E., Andersen, E. C., Null, B., White, K. P., Scott, M.P. (2001). Pattern of gene expression during Drosophila mesoderm development. Science. 293, 1629-33.
27. Garner, C. C., Nash, J. and Huganir, R. L. (2000). PDZ domains in synapse assembly and signalling. Trends Cell Biol. 10, 274-80.
28. progenitors in the Drosophila embryonic central nervous system. Novartis Found Symp. 237:139-51
29. Hakeda-Suzuki, S., Ng, J., Tzu, J., Dietzl, G., Sun, Y., harms, M., Nardine, T., Luo, L. and Dickson , B.J. (2002). Rac function regulation during Drosophila development. Nature 41, 438-442.
30. Nose, A., isshiki, T. and takeichi, M. (1998). Regional specification of muscle progenetors in Drosophila: the role of the msh homeobox gene. Development. 125, 215-23.
31. Regulation of actin assembly by SCAR/WAVE proteins. Biochem Soc Trans. 6, 1243-6.
32. Higgs, H. N., Pollard, T.D. (1999). Regulation of actin polymerization by Arp2/3 complex and WASp/Scar proteins. J Biol Chem. 46, 32531-4.
33. Rau, A., Buttgereit, D., Holz, A., Fetter, Doberstein, S. K., Paullulat, A., Staudt, N., Skeath, J., Michelson, A.M. and Renkawitz- Pohl, R. (2001). rolling pebbles (rols) is required in Drosophila muscle precursors for recruitment of myoblasts for fusion. Development 128, 5061-5073.
34. And Abmayr, S.M (2004). SNS: Adhesive properties, Localization requirement and ectodomain dependence in S2 cells and embryonic myoblasts. Mech. Dev. 121, 1455- 688.
35. Citovsky, V., Lee, L.Y., Vyas, S., Glick, E., Chen, M.H., Vainstein, A., Gafni, Y., Gelvin, S.B., and Tzfira, T. (2006). Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta. J Mol Biol 362, 1120-1131.
36. Machesky, L.M. and Gould, K.L. (1999). The Arp2/3 complex: a multifunctional actin organizer. Curr Opin Cell Biol. 1, 117-21.
37. Goley, E. D. and Welch, M.D., (2006). The ARP2/3 complex: an actin nucleator comes of age. Nat. Rev Mol Cell Biol. 10, 713-26.
38. Dottermusch, C., Verena, G., Klaus, E.S., Renkawitz-Pohl, R. And Onel, S.-F. The Drosophila Arf-GEF Schizo/Loner by the Arf- GAPs D-gitt or RhoGAP. MOD submitted.
39. Desai, C. J., garrity, P. A., Keshishian, H., Zipursky, S. L. and Zinn, K. (1999). The Drosophila SH2-SH3 adaptor protein Dock is expressed in embryonic axons and facilitates synapse formation by the RP3 motoneuron. Development. 126, 1527-1535.
40. Estrada, B., Maeland, A. D., Gisselbrecht, S. S., Bloor, J. W., Brown, N. H. and Michelson, A. M. (2007). The MARVEL domain protein, Singles Bar, is required for progression past the pre-fusion complex stages of myoblast fusion. Dev Biol 307, 328-39. Frasch, M. (1999). Controls in patterning and diversification of somatic muscles during Drosophila embryogenesis. Curr. Opin. Genetic. Dev. 9, 522-529.
41. Schäfer, G., (2008). The role of actin- regulators WASP and SCAR during Drosophila melanogaster muscle development. Philipps university of Marburg, " Ph. D thesis " .
42. Schäfer, G., Weber, S., Holz, A., Bogdan, S., Schumacher, S. Müller, A., Renkawitz-pohl, R. and Önel, S. F (2007). The Wiskott-Alrdich Van Embden, B., Ramos, H., Panchanathan, S., Newfeld, S.J., Kumar, S., and Tempe, A. Z. (Arizona State University; 2006). FlyExpress: an image-matching web-tool fo finding genes with overlapping patterns of Expression in Drosophila embryos. www.flyexpress.net.
43. Ho, H. Y., Rohatgi, R., Lebesohn, A. M., Le, M., Li, J., Gygi, S. P., and Kirschner, M. W. (2004). Toca-1 mediates Cdc42-dependent actin nucleation by activating the N-WASp- WIP complex. Cell 118, 203-16.
44. FuRMAS; Triggering myoblast fusion in Drosophila. Dev Dyn 238.1513-1525.
45. Martinez-Quiles, N., Rohatgi, R., Antón, I. M., Medina, M., Saville, S. P., Miki, H., Yamaguchi H., Takenawa, T., Hartwig, J. H., Geha. R. S., Ramesh, N. (2001). WIP regulates N-WASP-mediated actin polymerization and filopodium formation. Nat Cell Biol 3, 484-91.
46. Massarwa, R., Carmon, S., Shilo, B. Z and Schejter, .S. D. (2007). WIP/WASP-based actin polymerization machinery is essential for myoblas fusion in Drosophila. Dev Cell 12, 557-69.
47. Hu, C.D., Chinenov, Y., and Kerppola, T.K. (2002). Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell 9, 789-798.
48. Ruiz-Gomez, M., Coutts, N., Price, A., Taylor, M. V. and Bate, M. (2000). Drosophila dumbfounded: a myoblast attractant essential for fusion. Cell 102, 189-98.
49. Verma, R., Kovari, I., Soofi, A., Nihalani, D., Partie, K and Holzman, L.B. (2006). Nephrin ectodomain engagment results in Src kinase activation nephrin phosphorylation, Nck recruitment, and actin polymerization. J Clin Invest. 5, 1346-59.
50. Ruotsalainen, V., Patrakka, J., Tissari, P., Reponen, P., Hess, M., Kestilä, M., Holmberg, C., Salonen, R., Heikinheimo, M., Wartiovaara, J., Tryggvason, K., and Jalanko, H. (2000). Role of nephrin in cell junction formation in human nephrogenesis. Am J Pathol. 6, 1905-16.
51. Doberstein, S.K., Fetter, R.D., Mehta, A>Y> Goodman, C. S. (1997). Genetic analysis of myoblast fusion: blown fuse is required for progression beyond the prefusion complex. J.
52. Erickson, M. R., Galletta, B. J., and Abmayr, S.M. (1997). Drosophila myoblast city encodes a conserved protein that is essential for myoblast fusion, dorsal closure, and cytoskeletal organization. J Cell Biol. 138, 589- 603.
53. Machado, C., and Andrew, D. J. (2000). D- Titin: a giant protein with dual roles in chromosomes and muscles. J. Cell Biol. 151, 639-652.
54. The Drosophila HEM-2/NAP1 homolog KETTE controls axonal pathfinding and cytoskeletal organization. Genes Dev. 7, 863-73.
55. Kiyokowa, E., Hashimoto, Y., Kobayashi, S., Sugimura, H., Kurata, T. and Mastsuda, M. (1998). Activation of Rac1 by a Crk SH3- binding protein, DOCK 180. Genes Dev. 21, 3331-6.

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