Veränderung der subzellulären Lokalisation von Paxillin in ATP- und Bradykinin-stimulierten primären bovinen Osteoblasten

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 de...

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Bibliographic Details
Main Author: Silber, Ann-Sophie Luise
Contributors: Jones, D.B. (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2015
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The comprehensive roles of the skeleton rely on the mechanosensitive and mediator-responsive cellular response to external stimuli. Mechanical strain, growth, inflammation and carcinogenesis demand morphological adaptations of bone tissue, which are based on the adaptability of the cyto-skeleton. This thesis reports changes in the subcellular distribution of the fo-cal adhesion (FA) protein paxillin and intracellular calcium levels upon sti-mulation of cultured primary bovine osteoblasts (pOB) with extracellular ATP (100 µM) or bradykinin (1 µM). Stimulation with ATP or bradykinin induced a rapid redistribution of eGFP-tagged paxillin into newly formed, transient clusters at the plasma membrane, which was preceded by a significant rise in intracellular calcium concentrations. Using phase contrast and flu-orescence microscopy as well as TIRF microscopy, these paxillin-containing clusters were observed at the cell periphery. They occurred 100 seconds af-ter the addition of the ligands and vanished after 25 minutes. Using Fura-2AM, both ligands produced a significant calcium elevation within 20 se-conds, which clearly preceded the changes of the cytoskeleton. FRAP ana-lysis confirmed the rapid and nearly complete exchange of eGFP-tagged paxillin at stable, mature FAs, indicating the high mobility of paxillin and its fast recruitment to FAs. The latency between the maximal calcium peak and the subsequent cytoskeletal changes including the formation of paxillin-containing, membrane associated aggregates suggests complex signal cascades.