Exploring fibronectin's integrin binding domain effects on lung fibroblast integrin specificity and downstream phenotypic differences

Fibronectin;Integrin;Mechanobiology;Matrix biology;Extracellular matrix

Bachman, Haylee N. ; Finn, M. G. Chemistry and Biochemistry Williams, Loren Barry, Bridgette Lam, Wilbur ; Finn, M. G.

University:Georgia Institute of Technology

Department:Chemistry and Biochemistry

关键词: Fibronectin;    Integrin;    Mechanobiology;    Matrix biology;    Extracellular matrix;   

Others  :  https://smartech.gatech.edu/bitstream/1853/60669/1/BACHMAN-DISSERTATION-2017.pdf

美国|英语

来源: SMARTech Repository

【 摘 要 】

Recombinant proteins which mimic fibronectin’s (Fn’s) integrin binding domain in conformationally stable and unfolded states are investigated to explore integrin specificity and downstream phenotypic differences on lung fibroblasts. The recombinant protein expression and purification systems are redesigned for optimization and ease of isolation. Protein purification is validated and protein function is confirmed. These recombinant proteins are then used to explore biological function such as differential integrin engagement and focal adhesion associated signaling proteins. The “integrin-switch” behavior is characterized, focal adhesion quality is investigated, and force-mediated focal adhesion components FAK and Src are observed for phosphorylation in combination with recombinant variant Fn fragments. Lung fibroblast phenotype is examined based on the conformational bias of Fn’s integrin binding domain as well. Evalutation of cell contractility is explored via cell shape and size. Cell proliferation and etabolism are assessed to determine differences based on Fn fragment conformation presentation. The myfibroblast indicator of MRTF nuclear translocation is calculated to define how myo-like lung fibroblasts are with differential integrin engagement. These findings suggest that by mimicking stable vs unfolded conformations of Fn’s IBD different integrins are engaged and downstream cell behavior is affected.There appears to be a temporal relationship between lung fibroblasts and their substrate exposure which may cause phenotype changes that lead to myofibroblast dysregulation.