title: Dynamics and Interactions of Individual Proteins in the Membrane of Single, Living Cells.
abstract: Total internal reflection fluorescence (TIRF) microscopy is a powerful technique for interrogating protein dynamics in the membranes of living single cells. Receptor-ligand interactions are of particular interest for improving our understanding of cell signaling networks in a variety of applications. Here, we describe methods for fluorescently labeling individual receptors and their ligands, conducting single-molecule TIRF microscopy of receptors and ligands in single, living cells, and importantly, performing image analysis on the resulting time sequence of images to extract quantitative dynamics. While we use Toll-like receptor 4 and its ligand lipopolysaccharide as a specific example, the methods are general and readily extendable to other receptor-ligand systems of importance in cellular biology.
authors.affiliation: Sandia National Laboratories, Bioenergy and Defense Technologies, 5800, Albuquerque, NM, 87185, USA.
authors.forename: Stephen
authors.initials: S
authors.lastname: Anthony
authors.affiliation: Sandia National Laboratories, Bioenergy and Defense Technologies, 5800, Albuquerque, NM, 87185, USA.
authors.forename: Amanda
authors.initials: A
authors.lastname: Carroll-Portillo
authors.affiliation: Sandia National Laboratories, Bioenergy and Defense Technologies, 5800, Albuquerque, NM, 87185, USA. jatimli@sandia.gov.
authors.forename: Jerilyn
authors.initials: J
authors.lastname: Timlin
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D061848:Optical Imaging / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods*; D012984:Software; D051197:Toll-Like Receptor 4 / Q000032:analysis / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D018796:Immunoconjugates; D008565:Membrane Proteins; D051197:Toll-Like Receptor 4
keywords: Fluorescence microscopy; Single-molecule methods; Single-particle tracking; Superresolution