Novel Methods for Studying Ras/Erk MAP Kinase Signaling in Developing T Cells

FRET;intracellular staining;lentivirus;MAP Kinase;thymocytes

Green, Harry Miguel ; Alberola-Ila, Jose

University:California Institute of Technology

Department:Biology

关键词: FRET;    intracellular staining;    lentivirus;    MAP Kinase;    thymocytes;   

Others  :  https://thesis.library.caltech.edu/4234/1/HGdissertation.pdf

美国|英语

来源: Caltech THESIS

【 摘 要 】

The Ras/Erk MAPK pathway has been shown to be important in multiple developmental contexts.The development of T cells in the thymus is one such developmental system.Thymocytes undergo positive and negative selection, processes by which they are "chosen" for their ability to recognize MHC molecules loaded with peptide on the surface of cells, but only to react when the peptide is foreign.The Ras/Erk cascade has been shown to be indispensable during the onset of positive selection, but the mechanism of Erk signaling in this process is unknown.In addition, it is unclear if the Ras/Erk cascade is involved in the differentiation phase of positive selection called CD4/CD8 lineage determination, where thymocytes either become CD4+ or CD8+ T cells.Furthermore, Erk signaling has been shown to be activated during negative selection, but seems dispensable.In this thesis, we describe novel methods for analyzing Erk signaling by applying new technologies to gain a different perspective on Erk signaling in thymocytes during selection.To this end, we have utilized a technique of intracellular staining to obtain data for single-cell Erk activation in the context of a population of fixed thymocytes.We also pursued the development of FRET-based, genetically-encoded intracellular sensors of Erk activity that could be applied to the analysis of Erk signaling in live thymocytes in vivo.To examine the involvement of Ras/Erk signaling during CD4/CD8 lineage determination, we applied a recently described method of lentiviral transgenesis to examine dose-dependent effects of a dominant negative form of Mek, the Erk MAPK kinase, in a single mouse generation.These studies have yielded insights into Erk signaling events and advanced the development of novel techniques to examine signaling during thymocyte selection.