UBC Theses and Dissertations
In vivo investigation of talin, tensin, and integrin-linked kinase dynamics at stable cell-extracellular matrix adhesions Czerniecki, Stefan
Cell-extra cellular matrix (ECM) adhesion through the integrin family of receptors is required for metazoan development, and throughout adult life. Elucidating the mechanisms that regulate this adhesion is fundamental to understanding how animals create and maintain tissue architecture. Modulating adhesion assembly and disassembly is one of the key ways in which adhesion strength and integrity is regulated. We concentrate on analyzing the dynamics of three important components of the integrin adhesion complex (IAC), talin, tensin, and ILK, to determine how they function as mechano-sensory components of cell-ECM adhesions in the context of a living, multicellular organism, Drosophila melanogaster. We utilize fluorescently-tagged proteins under conditions of altered mechanical force, combined with a specialized fluorescence recovery after photobleaching (FRAP) protocol, to examine the dynamics of talin, tensin, and ILK. We subsequently use advanced mathematical modeling to gain mechanistic insight into how protein turnover is modified by tensile force. Furthermore, we attempt to clarify the role of key talin domains in mechanosensation, using FRAP and Drosophila homologs of previously characterized talin mutations, under conditions of altered force. The results outlined in this work show that talin mobility is directly regulated by force in an intact, complex organism at sites of stable adhesion between integrins and the ECM. Moreover, the results indicate that the mobility change due to increased force is a robust process, and not easily disrupted by mutating talin domains. Changes in talin dynamics when force is reduced is an active process, and is dependent on both the physical linkage of talin to integrin, and the ability of talin to auto-inhibit. Furthermore, studies of talin, tensin, and ILK turnover with high-temporal resolution uncover the intricacies of adhesion regulation in response to changing environmental conditions, with talin primarily regulated on the level of recycling, tensin regulated by a mix of both recycling and binding, and ILK regulated through control of binding.
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