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Analysis of the role of talin as a mechanosensor at the cell adhesions to the extracellular matrix in Drosophila melanogaster

University of British Columbia

Cells in multicellular organisms are arranged in complex three-dimensional patterns. To achieve such complexity, cells must form adhesive contacts with the extracellular matrix (ECM). The most common adhesion receptors that mediate cell-ECM adhesions are the integrins. A large cytoplasmic network of proteins, namely the integrin adhesion complex (IAC) is recruited to the site of adhesions. Regulated assembly and disassembly, or turnover, of the IAC is essential for dynamic cell movements and tissue maintenance. In this project, I sought to investigate the role of mechanical force on the turnover of talin, a core component of the IAC and an essential linker between integrins and the actin cytoskeleton. To investigate the turnover of talin in vivo, I performed fluorescence recovery after photobleaching (FRAP) on the myotendionous junctions (MTJs) in live Drosophila embryos and larvae. I used temperature sensitive mutants to alter the force acting on the MTJs. To better understand talin turnover, I collaborated with people from Dan Coombs’ lab (Department of Mathematics, UBC) to develop a mathematical model for the turnover of cytoplasmic adhesion proteins. This model is parametrized by four rate constants: talin binding on and off the adhesion complex at the plasma membrane, talin delivery to the plasma membrane due to the assembly of the IAC and talin removal from the plasma membrane due to the disassembly of the IAC. I hypothesized that changes in force would affect talin turnover and certain functional domains in talin would be required for mechanosening at the MTJs. I used targeted point mutations in the functional domains of talin to investigate their role in mechanosensing. Consistent with my hypothesis, I found out that disrupting functional domains in talin either abrogates or severely affect the ability of talin to respond to changes in force. First, these results provide direct evidence on how force is sensed at the adhesion complex. Secondly, this is the first in vivo study where four rate constants are used to characterize the turnover of a cytoplasmic protein.

Text

2015-09-30

Attribution-NonCommercial-NoDerivs 2.5 Canada

http://hdl.handle.net/2429/52658

Cell and Developmental Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/2.5/ca/