title: Real-Time Single Molecule Visualization of SH2 Domain Membrane Recruitment in Growth Factor Stimulated Cells.
abstract: In the last decade, single molecule tracking (SMT) techniques have emerged as a versatile tool for molecular cell biology research. This approach allows researchers to monitor the real-time behavior of individual molecules in living cells with nanometer and millisecond resolution. As a result, it is possible to visualize biological processes as they occur at a molecular level in real time. Here we describe a method for the real-time visualization of SH2 domain membrane recruitment from the cytoplasm to epidermal growth factor (EGF) induced phosphotyrosine sites on the EGF receptor. Further, we describe methods that utilize SMT data to define SH2 domain membrane dynamics parameters such as binding (τ), dissociation (kd), and diffusion (D) rates. Together these methods may allow us to gain greater understanding of signal transduction dynamics and the molecular basis of disease-related aberrant pathways.
authors.affiliation: Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, CT, 06030, USA. mbiod@nus.edu.sg.|Mechanobiology Institute, National University of Singapore, Singapore, Singapore. mbiod@nus.edu.sg.
authors.forename: Dongmyung
authors.initials: D
authors.lastname: Oh
mesh_terms: D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism* / Q000494:pharmacology; D008856:Microscopy, Fluorescence / Q000379:methods; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D021381:Protein Transport; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000072760:Single Molecule Imaging / Q000379:methods*; D014162:Transfection; D018909:src Homology Domains
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; D011506:Proteins
keywords: Diffusion rate; Dissociation rate; Membrane trafficking; SH2 domain; Single molecule; Total internal reflection fluorescence microscopy (TIRFM)