Fluorescence resonance energy transfer-based subcellular visualization of FAK/Src activation by chemical and mechanical environment

Live-cell imaging;fluorescence resonance energy transfer (FRET);Biosensors;Src;Focal Adhesion Kinase (FAK);Lipid rafts;Extracellular Matrix (ECM);Integrins

Seong, Ji-Hye

关键词: Live-cell imaging;    fluorescence resonance energy transfer (FRET);    Biosensors;    Src;    Focal Adhesion Kinase (FAK);    Lipid rafts;    Extracellular Matrix (ECM);    Integrins;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/42185/Ji-Hye_Seong.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

In response to environmental cues, neural circuits are maintained and rewired by growth cone dynamics, which are regulated by cytoskeleton reorganization and focal adhesion dynamics. Src and focal adhesion kinase (FAK) are key signaling molecules involved in these processes during growth cone dynamics and cell migration, and furthermore the progression of diseases such as cancer. Upon various chemical and mechanical environmental factors, Src and FAK can be localized and activated at plasma membrane, which contains different microdomains called lipid rafts. In this thesis, specific Src/FAK biosensors based on fluorescence resonance energy transfer (FRET) were developed and further targeted to subcompartments of plasma membrane to visualize local Src/FAK signaling events in live cells with high spatiotemporal resolution.Utilizing the subcellular-targeted Src biosensors, I found two distinct Src populations at different microdomains of plasma membrane. In response to chemical stimulations, the activation of Src population at lipid rafts was slower and weaker compared to that outside lipid rafts. In contrast, FAK biosensors targeted to different membrane microdomains revealed that FAK activation mainly occurs at lipid rafts in response to both growth factors and integrin clustering. Furthermore, I found the differential signaling hierarchy of Src/FAK complex at lipid rafts in response to growth factors and integrin clustering, which may contribute to different modes of cellular responses to various physiological stimulations.In addition to chemical stimulations, this thesis also presents how FAK activity is regulated by mechanical environment. FAK activation on fibronectin was more dependent on surface rigidity and intracellular tension than on collagen, because integrin activation by fibronectin, but not by collagen, is tension-dependent. These results suggest that outside mechanical signals can be differently translated inside cells through different interactions between specific extracellular molecules and integrin subtypes.In summary, this thesis can advance our in-depth understanding on how Src/FAK signaling is regulated at microdomains of plasma membrane. The integration of FRET-based biosensors and subcellular targeting signals can be unique and powerful tools to study accurate signaling events at the subcellular level in live cells. This thesis also provides valuable insights on how Src/FAK signaling is regulated upon various chemical and mechanical factors, which will help us to understand growth cone dynamics and cell migration, as well as to develop effective therapeutic tools to treat diseases related to neuronal systems and cancer.