title: Quantifying the Assembly of Multicomponent Molecular Machines by Single-Molecule Total Internal Reflection Fluorescence Microscopy.
abstract: Large, dynamic macromolecular complexes play essential roles in many cellular processes. Knowing how the components of these complexes associate with one another and undergo structural rearrangements is critical to understanding how they function. Single-molecule total internal reflection fluorescence (TIRF) microscopy is a powerful approach for addressing these fundamental issues. In this article, we first discuss single-molecule TIRF microscopes and strategies to immobilize and fluorescently label macromolecules. We then review the use of single-molecule TIRF microscopy to study the formation of binary macromolecular complexes using one-color imaging and inhibitors. We conclude with a discussion of the use of TIRF microscopy to examine the formation of higher-order (i.e., ternary) complexes using multicolor setups. The focus throughout this article is on experimental design, controls, data acquisition, and data analysis. We hope that single-molecule TIRF microscopy, which has largely been the province of specialists, will soon become as common in the tool box of biophysicists and biochemists as structural approaches have become today.
authors: University of Iowa Carver College of Medicine, Iowa City, IA, United States.
authors: E M
authors: EM
authors: Boehm
authors: University of Iowa Carver College of Medicine, Iowa City, IA, United States.
authors: S
authors: S
authors: Subramanyam
authors: Faculty of Science, Cairo University, Giza, Egypt. Electronic address: moh.kar.ghoneim@cu.edu.eg.
authors: M
authors: M
authors: Ghoneim
authors: University of Iowa Carver College of Medicine, Iowa City, IA, United States. Electronic address: todd-washington@uiowa.edu.
authors: M Todd
authors: MT
authors: Washington
authors: University of Iowa Carver College of Medicine, Iowa City, IA, United States. Electronic address: maria-spies@uiowa.edu.
authors: M
authors: M
authors: Spies
mesh_terms: D000465:Algorithms; D046911:Macromolecular Substances / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods*; D009696:Nucleic Acids / Q000737:chemistry; D060066:Protein Interaction Maps; D000072760:Single Molecule Imaging / Q000379:methods*
chemical_list: D046911:Macromolecular Substances; D009696:Nucleic Acids
keywords: Colocalization single-molecule imaging; Competitive and noncompetitive inhibition; Kinetic event resolving algorithm; Protein–protein and protein–nucleic acid interaction; Single-molecule fluorescence; Total internal reflection fluorescence microscopy