FRET-BASED BIOSENSORS TO ELUCIDATE EXTRACELLULAR SIGNAL-REGULATED KINASE (ERK) DYNAMICS

Biosensors;Signal transduction pathways;Biomedical Engineering

Hamidzadeh, Archer R.Zhang, Jin ;

Johns Hopkins University

关键词: Biosensors;    Signal transduction pathways;    Biomedical Engineering;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/38123/HAMIDZADEH-THESIS-2015.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Proper cell functions are dependent on the precise regulation of transduction events to relay external environmental cues into the cell for processing. The propagation and regulation of the signal involves a diverse array of enzymes and molecules with distinct functions and differences in spatio-temporal regulation. Further understanding cell signaling dynamics will continue to provide a knowledge basis for developing disease therapies. While an active field of research, a great deal of information has remained elusive with the previously used tools to monitor such pathways either through discrete snapshots or analyses of single components of signaling pathways outside of their natural biological context. To address these limitations, FRET-based biosensors provide a powerful means to dissect the complexities of signaling pathways in a real-time manner in live cells.The first objective is to optimize the previously developed ERK Activity Reporter, EKAR, by examining the dependence of its dynamic range on fluorophore orientation. To alter relative fluorophore orientations, variations of EKAR were developed with reversed orders of fluorescent proteins.Reversing the order of a previously optimized yellow-cyan EKAR led to a cyan-yellow EKAR with an improved dynamic range. Moreover, a closer examination of the previously optimized EeVee-linker, which was believed to render FRET-based biosensors solely distance-dependent, reveals that the FRET efficiency is likely dependent on FP orientation in EeVee-linker based biosensors. This conclusion for EKAR can likely be extended for further improvements of other FRET-based biosensors with the EeVee linker. The second objective is to expand the versatility of the EKAR biosensor for co-imaging capabilities, which allow for more precise correlations between multiple activity readouts on a single-cell level. Utilizing EKAR color variants, a co-imaging strategy was employed to allow for imaging two sensors with spectrally distinct FRET-donor fluorescent proteins (FP) and a common FRET-acceptor FP. This strategy allowed for simultaneously tracking ERK activity in multiple subcellular compartments, monitoring both ERK and Protein kinase A (PKA) on a single-cell level, and for the development of a novel single-chain biosensor capable of monitoring ERK and Ras activity in the plasma membrane.Continued investigations will provide powerful tools to elucidate signaling dynamics with stronger precision.