Visualization of cellular signaling by fluorescence resonance energy transfer in response to biochemical and mechanical microenvironments

Biochemical and mechanical microenvironment have a great impact on a variety of cellular processes including cell adhesion and migration. While substantial progress has been made toward the understanding of how these factors regulate many cellular functions, it remains elusive on the molecular mechanism by which stem cells perceive such biochemical/mechanical cues to coordinate signaling network to determine their behavior. Hence, visualization of the intracellular signals is a challenge of major importance for in-depth understanding of the relationship between biochemical/mechanical cues and various cellular responses. In this thesis, I have focused on Ca2+, cAMP-dependent protein kinas A (PKA), and focal adhesion kinase (FAK) signaling in human mesenchymal stem cells utilizing the genetically-encoded biosensors that are based on fluorescence resonance energy transfer (FRET). FRET technology is a powerful tool to detect and visualize protein-protein interactions and enzymatic activities in living cells with high spatiotemporal resolution. In this thesis, I show that substrate rigidity regulates the spontaneous Ca2+ oscillations which are mediated by RhoA pathway. Using optical laser tweezers in combination with the FRET Ca2+ sensors targeting the cytoplasm and endoplasmic reticulum, it reveals how Ca2+ signaling at the plasma membrane and ER membrane channels can be differently regulated by mechanical stimulation, in particular, distinct roles between active actomyosin contractility and passive cytoskeleton supports. In addition to these Ca2+ signals, I show that agonist-induced PKA activation is regulated by the different magnitude of substrate rigidity, which further reveals to mediate endocytosis of β2-adrenergic receptors in a microtubule-dependent manner. These findings appear to be very valuable due to cross-talk between Ca2+ and PKA during cell adhesion and migration. Moreover, this thesis shows that Ca2+ and FAK signals are differently activated in response to substrate rigidity at plasma membrane microdomains in the adhesion process. These results suggest that biochemical/mechanical factors have a great influence on various cellular signals such as Ca2+, PKA and FAK through various mechanisms. In summary, the integration of live cell imaging and FRET based biosensors capable of targeting the different subcellular compartments can provide more accurate cellular information in living cells. This thesis will also advance our in-depth understanding on how biochemical and mechanical microenvironment affect various cellular signals and their behavior.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Neuroscience

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2013

Author dc:creator

• Kim, Tae Jin

Contributors dc:contributor

• Wang, Yingxiao
• Leckband, Deborah E.
• Wang, Ning
• Kong, Hyun Joon

• Fluorescence resonance energy transfer (FRET)
• Live-cell imaging
• Cell Signals
• Microenvironment

Statement dc:rights

• Copyright 2013 Tae Jin Kim

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/45646

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/45646