Single-Molecule Photoactivation and Localization of Signaling Complexes in T cells

Abstract

In T cells, membrane receptor and ligand engagement initiates a signaling cascade. Ligand binding is relayed through specific membrane receptors, protein tyrosine kinases, and critical adaptors to regulate downstream activation of transcription factors, cytokine production and cell proliferation. The membrane segregation and relocalization of these signaling components display highly dynamic protein networks in response to distinct stimulations. However, the membrane-protein and protein-protein interactions involved in the formation of signaling complexes are still poorly understood. We are currently using a high-resolution imaging technique to visualize the early stage of signal transduction events at the single-molecule level with photoactivatable or photoconvertable fluorescent markers. The photoactivation and localization of T-cell signaling components allow measurements of molecular spatial and temporal correlation in response to different stimulations. With dual-color application, molecular correlation functions will enhance the understanding of the protein-protein interactions in signaling complexes.