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Single-Molecule Studies of Actin Assembly and Disassembly Factors

Authors
  • Smith, Benjamin A.
  • Gelles, Jeff
  • Goode, Bruce L.1, 2, 3
  • 1 Department of Biochemistry, Brandeis University
  • 2 Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University
  • 3 Present address: Biogen Idec
Type
Book
Journal
G Protein Coupled Receptors - Structure
Publisher
Elsevier BV
Publication Date
Jan 01, 2014
Volume
540
Pages
95–117
Identifiers
DOI: 10.1016/j.cell.2007.02.001
ISBN: 978-0-12-397924-7
Source
Elsevier
Keywords
License
Unknown

Abstract

The actin cytoskeleton is very dynamic and highly regulated by multiple associated proteins in vivo. Understanding how this system of proteins functions in the processes of actin network assembly and disassembly requires methods to dissect the mechanisms of activity of individual factors and of multiple factors acting in concert. The advent of single-filament and single-molecule fluorescence imaging methods has provided a powerful new approach to discovering actin-regulatory activities and obtaining direct, quantitative insights into the pathways of molecular interactions that regulate actin network architecture and dynamics. Here we describe techniques for acquisition and analysis of single-molecule data, applied to the novel challenges of studying the filament assembly and disassembly activities of actin-associated proteins in vitro. We discuss the advantages of single-molecule analysis in directly visualizing the order of molecular events, measuring the kinetic rates of filament binding and dissociation, and studying the coordination among multiple factors. The methods described here complement traditional biochemical approaches in elucidating actin-regulatory mechanisms in reconstituted filamentous networks.

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