The 3D architecture of cell adhesion molecules regulates calpain-mediated turnover of focal adhesion complex

  • 張 淑淨

Student thesis: Master's Thesis


Focal adhesions are large dynamic protein complexes which are close to the plasma membrane (within 15 nm) Focal adhesions serve as the mechanical linkages between the cytoskeleton and the extracellular matrix and as a biochemical signaling hub to concentrate and direct numerous signaling proteins at the sites of integrin binding and clustering The coordinated and dynamic regulation of focal adhesion is required for cell migration including assembly and degradation Degradation or turnover of focal adhesion is the major event in the rear end of a migratory cell which is in a Ca2+/calpain-dependent proteolysis In addition calpain has been shown to regulate the turnover of focal adhesion by proteolysis of multiple focal adhesion related proteins such as FAK Paxillin Vinculin and Talin The organization of focal adhesion proteins indicates a composite of several laminar architectures with spatial and functional compartments that mediate the interdependent functions of focal adhesion The effects of Ca2+ signal on focal adhesion turnover have been shown but most of them have not shown the detailed space-time cascades of focal adhesion turnover in real time The aim of this study is to investigate how a Ca2+ signal induces cascades of focal adhesion turnover in living cell The results of TIRFM images showed Ca2+ induces different processes of each focal adhesion molecule In addition three calpain-resistant mutant focal adhesion molecules were used to clarify the interactions between focal adhesion molecules The results showed Vinculin is difficult to be degraded and not be significantly affected by calpain-resistant mutanted focal adhesion molecules The 3D fluorescence imaging was performed to confirm the relationship between architecture and degradation of focal adhesion The results showed architecture of focal adhesion is related to the trend of degradation of focal adhesion molecules Traditional approaches in the study of focal adhesion dynamics have been via pharmacological stimulation or genetic manipulation However these methods have poor temporal and spatial precision An optogenetics is the combination of genetics and optics to control well-defined events within specific cells of living tissue in real time Optogenetic stimulation approach that uses channelrhodopsin-2 (ChR2) has been developed for providing more precise and targeted stimulation effect on cells ChR2 is a directly light-switched cation-selective ion channel which absorbs blue light with an absorption maximum of 480 nm Then this channel opens rapidly to generate a large degree of permeability for monovalent and divalent cations conducting H+ Na+ K+ and Ca2+ ions The results showed that ChR2 can be used to cause global and local Ca2+ influxes Taken together this study suggests that the 3D architecture of focal adhesion plays an important role in Ca2+-mediated focal adhesion turnover Therefore optogenetic stimulation of ChR2 can be used to operate detailed focal adhesion turnover
Date of Award2014 Jun 30
Original languageEnglish
SupervisorWen-Tai Chiu (Supervisor)

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