NEW TECHNOLOGIES FOR LIVE CELL FLUORESCENCE IMAGING OF POST-TRANSLATIONAL MODIFICATIONS

Biosensors;Post-translational modifications;Fluorescent Proteins;Signal Transduction;Cell Signaling;Biomedical Engineering

Ross, Brian LouisKuo, Scot ;

Johns Hopkins University

关键词: Biosensors;    Post-translational modifications;    Fluorescent Proteins;    Signal Transduction;    Cell Signaling;    Biomedical Engineering;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/60099/ROSS-DISSERTATION-2018.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Post translational modifications (PTMs) of proteins serve critical roles in both signal transduction as well as gene regulation. Phosphorylation is an important means by which signals are transmitted, processed, and amplified within a cell, while methylation is a critical regulator of chromatin organization mediated by histone proteins. Thespatial distribution and temporal dynamics of these PTMs must be tightly controlled to allow for proper functioning. In signaling pathways, distinct spatiotemporal patterning of signaling activities is a critical means by which cells use the same molecular components to regulate diverse functionalities. In gene regulation, precise spatial organization of histone methylation allows for the proper distributions of heterochromatin and euchromatin, controlling which parts of the genome are transcriptionally active or repressed.In this dissertation, we discuss three new genetically encodable fluorescence technologies to expand the information we can obtain about the spatiotemporal dynamics of PTMs in real time in live cells. First, we discuss a new suite of genetically encodable fluorescent biosensors that are both single-color and ratiometric. These FLuorescent Anisotropy Reporters (FLARE) are based on homo-FRET, and they allow for multiparameter imaging of cell signaling activities, such as kinase activity and second-messengers. We then present a panel of biosensors called Fluorescent fLuctuation INcrease by Contact (FLINC) sensors that allow for the creation of sub-diffraction limit resolution maps of kinase activity. They were then used to map highly localized signaling microdomains of the Protein Kinase A (PKA) pathway in the plasma membrane. Finally, we describe a new fluorescent probe for superresolution mapping of tri-methylated histone 3 lysine 9 (H3K9me3), a critical histone modification for regulating the formation of heterochromatin in the nucleus. By engineering a mutant of the chromobox homolog protein 1 (CBX1) with high affinity for H3K9me3 and fusing it with a photoactivatable fluorescent protein, we demonstrate the ability to create sub-diffraction limit resolution maps of H3K9me3 in live cells.