Development of Multiplexable Biosensors to Quantify the Spatiotemporal Dynamics of Rho GTPases and Protein Kinases in the Same Living Cell Public Deposited

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  • March 22, 2019
Creator
  • Hsu, Chia-Wen
    • Affiliation: Eshelman School of Pharmacy, Division of Chemical Biology and Medicinal Chemistry
Abstract
  • Cell motility is a highly dynamic and heterogenous cellular process regulated by the coordination of multiple Rho GTPases, Src family kinases, and the mitogen-activated protein kinase (MAPK) cascades. However, it has been difficult to monitor more than two protein activities in the same cell due to the overlapping spectra of current biosensors and biological perturbations at high biosensor concentrations. Dye-based biosensors, which rely on an affinity scaffold that binds only to the activated conformation of the endogenous targets and an environment-sensing dye that changes its fluorescence properties to report the specific binder-target interactions, possess great potential to monitor multiple endogenous targets in the same cell. Here, I created novel environment-sensing dyes and exploited novel affinity scaffolds to develop multiplexable dye-based biosensors capable of quantifying the spatiotemporal dynamics of multiple Rho GTPases and protein kinases in the same cell. Src protein kinase is an upstream regulator of the Cdc42 GTPase. The coordination of Cdc42 and Src at the leading edge has not been well characterized due to lack of multiplexable biosensors to monitor Cdc42 and Src activities in the same living cell. Therefore, I developed novel near infrared merocyanine dyes and a red ratiometric merocyanine dye with an intrinsic ratiometric response that can be used to construct multiplexible biosensors. The relative timing and the subcellular localization of active Cdc42 and Src during leading edge dynamics and during pinocytosis were revealed using the new dyes. Src also plays an important role in the MAPK-mediated cell motility. However, the precise roles of MAPKKs and MAPKs at the leading edge remain poorly characterized due to the lack of sensitive biosensors for each target. By taking advantage of the specific interactions between MAPKKs and MAPKs, I developed the first substrate-based biosensor designs to report the activity of endogenous MEK1/2 and MKK3/6. I also developed a sensitive ERK1/2 biosensor based on artificial binders through collaborations with the Plűckthun group. This work will provide a foundation to study the crosstalk between Rho GTPases, Src family kinases and the MAPK cascades via multiplexed live cell imaging.
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  • In Copyright
Advisor
  • Hahn, Klaus
Degree
  • Doctor of Philosophy
Graduation year
  • 2012
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