Date of Graduation
School of Medicine
Microbiology, Immunology, and Cell Biology
Focal adhesions are specialized cell contact sites of distinct molecular composition and structure that bridge the actin cytoskeleton to the extracellular matrix and provide for efficient bidirectional transmission of biochemical and mechanical signals between the intra- and extracellular compartment. Many proteins within the focal adhesion have been discovered to be an integral component of the adhesion structure and function; however, a key molecule in the organization and physiological activity of focal adhesions is focal adhesion kinase (FAK). FAK is a nonreceptor tyrosine kinase that is essential for cell processes including cell migration, growth, and survival. Due to its connection between the cytoskeleton and extracellular matrix, FAK has been proposed to be a key component of integrin downstream signaling that regulates the organization of actin for transduction of cellular forces from inside to outside of the cell. While many studies have focused on determining FAK's role in sensing mechanical forces and regulating contractile signaling pathways, very few studies have attempted to determine the role of FAK in cell contraction through direct measurement of cellular tension. Therefore, the role of FAK in fibroblast and endothelial cell contractility was determined.;To investigate the role of FAK in endothelial cell tension and monolayer permeability, a stable FAK knockdown human pulmonary microvessel endothelial cell line (FAK-KD) was generated. Knockdown of FAK altered both cell morphology and actin distribution, and increased focal adhesion formation and VE-cadherin localization to cell-cell contacts. Measurement of tension produced by cells embedded within a three-dimensional (3-D) collagen matrix revealed that loss of FAK increased basal tension without alterations in basal myosin phosphorylation. Agonist-induced force was unaffected. However, loss of FAK enhanced endothelial monolayer barrier function. Thus, FAK is responsible for the balance between cellmatrix and cell-cell cohesion in order to regulate endothelial cell tension and monolayer permeability.;In order to determine the role of FAK in fibroblast cell contractility, FAK knockout (FAK-KO) mouse embryonic fibroblasts (MEFs) embedded in 3-D collagen gels were utilized. FAK null MEFs produced a decrease in basal tension and minimal agonist induced force compared to controls (FAK-WT). However, myosin II phosphorylation was comparable between FAK-KO and FAK-WT MEFs. Investigation of the collagen matrix revealed that FAKKO MEFs had an inability to organize their collagen matrix. Inhibition of FAK kinase activity or expression of FAK mutants revealed that FAK kinase activity was dispensable for tension generation. Thus, FAK localization to the focal adhesion was critical in the transmission of internal force to the collagen matrix resulting in cell contraction.;Collectively, these data show that FAK is an integral part in nonmuscle cellular tension. Although the loss of FAK altered tension generation differently in fibroblasts and endothelial cells, the differences in each cell's physiological function may explain why FAK regulates cell tension differently. Nevertheless, FAK is an important molecular player in focal adhesions facilitating the transduction of forces from inside to outside of the cell and may be a novel target in the development of treatments to control cell contractility.
Arnold, Kimberly M., "The role of focal adhesion kinase in nonmuscle cell contraction" (2013). Graduate Theses, Dissertations, and Problem Reports. 4949.