Imaging molecular interactions in living cells.
Hormones integrate the activities of their target cells through receptor-modulated cascades of protein interactions that ultimately lead to changes in cellular function. Understanding how the cell assembles these signaling protein complexes is critically important to unraveling disease processes, and to the design of therapeutic strategies. Recent advances in live-cell imaging technologies, combined with the use of genetically encoded fluorescent proteins, now allow the assembly of these signaling protein complexes to be tracked within the organized microenvironment of the living cell. Here, we review some of the recent developments in the application of imaging techniques to measure the dynamic behavior, colocalization, and spatial relationships between proteins in living cells. Where possible, we discuss the application of these different approaches in the context of hormone regulation of nuclear receptor localization, mobility, and interactions in different subcellular compartments. We discuss measurements that define the spatial relationships and dynamics between proteins in living cells including fluorescence colocalization, fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy. These live-cell imaging tools provide an important complement to biochemical and structural biology studies, extending the analysis of protein-protein interactions, protein conformational changes, and the behavior of signaling molecules to their natural environment within the intact cell.
Day
Richard N
RN
Department of Medicine, P.O. Box 800578, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA. rnd2v@virginia.edu
Schaufele
Fred
F
D000818:Animals; D002477:Cells / Q000737:chemistry; D006728:Hormones / Q000502:physiology*; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis*; D008856:Microscopy, Fluorescence; D011506:Proteins / Q000032:analysis*; D018160:Receptors, Cytoplasmic and Nuclear / Q000502:physiology; D013050:Spectrometry, Fluorescence / Q000379:methods*
D006728:Hormones; D008164:Luminescent Proteins; D011506:Proteins; D018160:Receptors, Cytoplasmic and Nuclear