Dokument

Zusammenfassung:
Die Funktionalität des Skelettsystems beruht auf der mechanosensitiven und mediatorresponsiven Anpassung des Knochengewebes an äußere Ereignis-se. Mechanische Kraftbeanspruchung, Wachstum, Entzündung und Karzi-nogenese erfordern morphologische Veränderungen des Gewebes, welche auf der Adaptivität der einzelnen Zellen beruhen. In dieser Arbeit wurde die Reaktion von primären bovinen Osteoblasten (pOB) auf eine Stimulation mit extrazellulärem ATP (100 µM) und Bradykinin (1 µM) hinsichtlich der subzel-lulären Verteilung des FA-Proteins Paxillin und Veränderungen im intrazellu-lären Kalziumspiegel untersucht. Unter ATP- oder Bradykinin-Stimulation kam es in eGFP-Paxillin exprimierenden pOB zur schnellen Umverteilung von Paxillin in transiente fokale Cluster mit zeitlich vorausgehendem Anstieg des intrazellulären Kalziumspiegels. Mittels Phasenkontrastmikroskopie, Flu-oreszenzmikroskopie und TIRF-Mikroskopie konnten 100 Sekunden nach der Stimulation der Osteoblasten Paxillin-haltige Aggregate in der Zellperipherie beobachtet werden, deren Entwicklung innerhalb von 25 Minuten rückläufig war. Behandlung mit beiden Mediatoren induzierte innerhalb von 20 Se-kunden, und somit vor den sichtbaren Zytoskelett-Veränderungen signifikan-te Kalziumanstiege, die über den Kalziumindikator Fura-2AM gemessen wur-den. Mittels FRAP-Technologie konnten zudem hohe Austauschraten von eGFP-markiertem Paxillin in stabilen, maturierten FA dokumentiert werden. Die schnellen, mehr-phasischen und nahezu kompletten Austauschprozes-se von Paxillin in reifen FA lassen auf eine hohe, dynamische Mobilität des Proteins innerhalb des Zytoplasmas schließen. Die Latenz zwischen den Veränderungen des Kalziumspiegels und der Entstehung von Paxillin-haltiger Membranstrukturen weist auf komplexe Signalkaskaden hin.

Bibliographie / References

1. Turner CE, Glenney JR, Burridge K (1990) Paxillin: a new vinculin- binding protein present in focal adhesions. J Cell Biol 111:1059-1068
2. Takahashi A, Camacho P, Lechleiter JD, Herman B (1999) Measure- ment of intracellular calcium. Physiol Rev 79:1089-1125
3. Rottner K, Hall A, Small JV (1999) Interplay between Rac and Rho in the control of substrate contact dynamics. Curr Biol 9:640-648
4. Yamato M, Okuhara M, Karikusa F, Kikuchi A, Sakurai Y, Okano T (1999) Signal transduction and cytoskeletal reorganization are required for cell detachment from cell culture surfaces grafted with a tempera- ture-responsive polymer. J Biomed Mater Res 44:44-52
5. Rees DA, Charlton J, Ataliotis P, Woods A, Stones AJ, Bayley SA (1989) Myosin regulation and calcium transients in fibroblast shape change, attachment, and patching. Cell Motil Cytoskeleton 13:112-122
6. Rozengurt E (2007) Mitogenic signaling pathways induced by G protein- coupled receptors. J Cell Physiol 213:589-602
7. Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509-544
8. Rahman S, Bunning RA, Dobson PR, Evans DB, Chapman K, Jones TH, Brown BL, Russell RG (1992) Bradykinin stimulates the production of prostaglandin E2 and interleukin-6 in human osteoblast-like cells. Biochim Biophys Acta 1135:97-102
9. Yu H, Ferrier J (1994) Mechanisms of ATP-induced Ca2+ signaling in osteoclasts. Cell Signal 6:905-914
10. Schaller MD (2001) Paxillin: a focal adhesion-associated adaptor pro- tein. Oncogene 20:6459-6472
11. Wagstaff SC, Bowler WB, Gallagher JA, Hipskind RA (2000) Extracellu- lar ATP activates multiple signalling pathways and potentiates growth factor-induced c-fos gene expression in MCF-7 breast cancer cells. Carcinogenesis 21:2175-2181
12. Yang TT, Cheng L, Kain SR (1996) Optimized codon usage and chro- mophore mutations provide enhanced sensitivity with the green flu- orescent protein. Nucleic Acids Res 24:4592-4593
13. Young MF, Kerr JM, Ibaraki K, Heegaard AM, Robey PG (1992) Structure, expression, and regulation of the major noncollagenous mat- rix proteins of bone. Clin Orthop Relat Res:275-294
14. Soon Seng Ng CL, Vincent Chan (2011) Experimental and numerical determination of cellular traction force on polymeric hydrogels. Inter- face focus, pp 777-791
15. Tippmer S, Bossenmaier B, Häring H (1996) Bradykinin induces tyro- sine phosphorylation in human foreskin fibroblasts and 293 cells trans- fected with rat B2 kinin receptor. Eur J Biochem 236:953-959
16. Wang M, Kong Q, Gonzalez FA, Sun G, Erb L, Seye C, Weisman GA (2005) P2Y nucleotide receptor interaction with alpha integrin mediates astrocyte migration. J Neurochem 95:630-640
17. Wang JW, Su W, Law YP, Lu CH, Chen YC, Wang JL, Chang HJ, Chen WC, Jan CR (2001) Mechanism of bradykinin-induced Ca(2+) mobi- lization in MG63 human osteosarcoma cells. Horm Res 55:265-270
18. Romer LH, Birukov KG, Garcia JG (2006) Focal adhesions: paradigm for a signaling nexus. Circ Res 98:606-616
19. Teranishi S, Kimura K, Nishida T (2009) Role of formation of an ERK- FAK-paxillin complex in migration of human corneal epithelial cells during wound closure in vitro. Invest Ophthalmol Vis Sci 50:5646-5652
20. Wongkhantee S, Yongchaitrakul T, Pavasant P (2008) Mechanical stress induces osteopontin via ATP/P2Y1 in periodontal cells. J Dent Res 87:564-568
21. Zaidel-Bar R, Milo R, Kam Z, Geiger B (2007) A paxillin tyrosine phos- phorylation switch regulates the assembly and form of cell-matrix ad- hesions. J Cell Sci 120:137-148
22. Wolfenson H, Bershadsky A, Henis YI, Geiger B (2011) Actomyosin-ge- nerated tension controls the molecular kinetics of focal adhesions. J Cell Sci 124:1425-1432
23. Weisman GA, Wang M, Kong Q, Chorna NE, Neary JT, Sun GY, González FA, Seye CI, Erb L (2005) Molecular determinants of P2Y2 nucleotide receptor function: implications for proliferative and inflamm- atory pathways in astrocytes. Mol Neurobiol 31:169-183
24. Yang HJ, Chen JZ, Zhang WL, Ding YQ (2010) Focal adhesion plaque associated cytoskeletons are involved in the invasion and metastasis of human colorectal carcinoma. Cancer Invest 28:127-134
25. Webb DJ, Donais K, Whitmore LA, Thomas SM, Turner CE, Parsons JT, Horwitz AF (2004) FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly. Nat Cell Biol 6:154-161
26. Verhoef PA, Estacion M, Schilling W, Dubyak GR (2003) P2X7 recep- tor-dependent blebbing and the activation of Rho-effector kinases, caspases, and IL-1 beta release. J Immunol 170:5728-5738
27. Yang WH, Chang JT, Hsu SF, Li TM, Cho DY, Huang CY, Fong YC, Tang CH (2010) Bradykinin enhances cell migration in human chondrosarcoma cells through BK receptor signaling pathways. J Cell Biochem 109:82-92
28. Zamir E, Katz BZ, Aota S, Yamada KM, Geiger B, Kam Z (1999) Molecular diversity of cell-matrix adhesions. J Cell Sci 112 ( Pt 11):1655-1669
29. Turner CE, Miller JT (1994) Primary sequence of paxillin contains putative SH2 and SH3 domain binding motifs and multiple LIM do- mains: identification of a vinculin and pp125Fak-binding region. J Cell Sci 107 ( Pt 6):1583-1591
30. Turner CE (2000) Paxillin interactions. J Cell Sci 113 Pt 23:4139-4140
31. Tokuda H, Kotoyori J, Oiso Y, Kozawa O (1994) Intracellular signaling mechanism of bradykinin in osteoblast-like cells: comparison with prostaglandin E2. Endocr J 41:189-195
32. Wang JH, Lin JS (2007) Cell traction force and measurement methods. Biomech Model Mechanobiol 6:361-371
33. Assistenzärztin in der Viszeralchirurgie in Bad Soden 12/2014
34. Wolff J (1892) Das Gesetz der Transformation der Knochen., Hirch- wild, Berlin
35. Totsukawa G, Yamakita Y, Yamashiro S, Hartshorne DJ, Sasaki Y, Matsumura F (2000) Distinct roles of ROCK (Rho-kinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fi- bers and focal adhesions in 3T3 fibroblasts. J Cell Biol 150:797-806
36. Tabellarischer Lebenslauf Persönliche Daten: Geburtsdatum: 19.04.1989 in Speyer, Rheinland-Pfalz Studium und Schule: Ab 01/02/2015
37. Zamir E, Geiger B (2001) Molecular complexity and dynamics of cell- matrix adhesions. J Cell Sci 114:3583-3590
38. Raisz LG (1999) Physiology and pathophysiology of bone remode- ling. Clin Chem 45:1353-1358
39. Rozengurt E (1998) Signal transduction pathways in the mitogenic response to G protein-coupled neuropeptide receptor agonists. J Cell Physiol 177:507-517
40. Vogel V (2006) Mechanotransduction involving multimodular proteins: converting force into biochemical signals. Annu Rev Biophys Biomol Struct 35:459-488
41. Tan L, Meyer T, Pfau B, Hofmann T, Tan TW, Jones D (2010) Rapid vinculin exchange dynamics at focal adhesions in primary osteoblasts following shear flow stimulation. J Musculoskelet Neuronal Interact 10:92-99
42. Soltoff SP, Avraham H, Avraham S, Cantley LC (1998) Activation of P2Y2 receptors by UTP and ATP stimulates mitogen-activated kinase activity through a pathway that involves related adhesion focal tyrosine kinase and protein kinase C. J Biol Chem 273:2653-2660
43. Zaidel-Bar R, Ballestrem C, Kam Z, Geiger B (2003) Early molecular events in the assembly of matrix adhesions at the leading edge of mig- rating cells. J Cell Sci 116:4605-4613
44. Riveline D, Zamir E, Balaban NQ, Schwarz US, Ishizaki T, Narumiya S, Kam Z, Geiger B, Bershadsky AD (2001) Focal contacts as mechano- sensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent me- chanism. J Cell Biol 153:1175-1186
45. Zheng LM, Zychlinsky A, Liu CC, Ojcius DM, Young JD (1991) Extracellular ATP as a trigger for apoptosis or programmed cell death. J Cell Biol 112:279-288
46. Schaller MD, Parsons JT (1995) pp125FAK-dependent tyrosine phosphorylation of paxillin creates a high-affinity binding site for Crk. Mol Cell Biol 15:2635-2645
47. Taniyama Y, Weber DS, Rocic P, Hilenski L, Akers ML, Park J, Hem- mings BA, Alexander RW, Griendling KK (2003) Pyk2-and Src-depen- dent tyrosine phosphorylation of PDK1 regulates focal adhesions. Mol Cell Biol 23:8019-8029
48. Wolfenson H, Lubelski A, Regev T, Klafter J, Henis YI, Geiger B (2009) A role for the juxtamembrane cytoplasm in the molecular dynamics of focal adhesions. PLoS One 4:e4304
49. Zaidel-Bar R, Itzkovitz S, Ma'ayan A, Iyengar R, Geiger B (2007) Functional atlas of the integrin adhesome. Nat Cell Biol 9:858-867
50. Snapp E (2005) Design and use of fluorescent fusion proteins in cell biology. Curr Protoc Cell Biol Chapter 21:Unit 21.24
51. Wolfenson H, Henis YI, Geiger B, Bershadsky AD (2009) The heel and toe of the cell's foot: a multifaceted approach for understanding the structure and dynamics of focal adhesions. Cell Motil Cytoskeleton 66:1017-1029
52. Spiering D, Hodgson L (2011) Dynamics of the Rho-family small GTPa- ses in actin regulation and motility. Cell Adh Migr 5:170-180
53. Rossow MJ, Sasaki JM, Digman MA, Gratton E (2010) Raster image correlation spectroscopy in live cells. Nat Protoc 5:1761-1774
54. Stricker J, Aratyn-Schaus Y, Oakes PW, Gardel ML (2011) Spatiotem- poral constraints on the force-dependent growth of focal adhesions. Bi- ophys J 100:2883-2893
55. Séror C, Melki MT, Subra F, Raza SQ, Bras M, Saïdi H, Nardacci R, Voisin L, Paoletti A, Law F, Martins I, Amendola A, Abdul-Sater AA, Ciccosanti F, Delelis O, Niedergang F, Thierry S, Said-Sadier N, La- maze C, Métivier D, Estaquier J, Fimia GM, Falasca L, Casetti R, Modjtahedi N, Kanellopoulos J, Mouscadet JF, Ojcius DM, Piacentini M, Gougeon ML, Kroemer G, Perfettini JL (2011) Extracellular ATP acts on P2Y2 purinergic receptors to facilitate HIV-1 infection. J Exp Med 208:1823-1834
56. Trache A, Meininger GA (2008) Total internal reflection fluorescence (TIRF) microscopy. Curr Protoc Microbiol Chapter 2:Unit 2A.2.1- 2A.2.22
57. Vatsa A, Semeins CM, Smit TH, Klein-Nulend J (2008) Paxillin localisa- tion in osteocytes--is it determined by the direction of loading? Bio- chem Biophys Res Commun 377:1019-1024
58. Yu H, Ferrier J (1993) ATP induces an intracellular calcium pulse in osteoclasts. Biochem Biophys Res Commun 191:357-363
59. Webb DJ, Brown CM, Horwitz AF (2003) Illuminating adhesion comple- xes in migrating cells: moving toward a bright future. Curr Opin Cell Biol 15:614-620
60. Yu J, Prado GN, Taylor L, Pal-Ghosh R, Polgar P (2002) Hybrid formation between the intracellular faces of the bradykinin B2 and angiotensin II AT1 receptors and signal transduction. Int Immunophar- macol 2:1807-1822
61. Sakata A, Ohmori T, Nishimura S, Suzuki H, Madoiwa S, Mimuro J, Kario K, Sakata Y (2014) Paxillin is an intrinsic negative regulator of platelet activation in mice. Thromb J 12:1

Das Dokument ist im Internet frei zugänglich - Hinweise zu den Nutzungsrechten