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The actin cytoskeleton plays a pivotal role in many cellular processes. Detailed analysis of the architecture of cellular actin networks provides valuable insight into the dynamic self-organization underlying these processes. In particular, since most of the actin turnover occurs at the tips of actin filaments, it is insightful to map the distribution of filament ends. Here we report a method for differentially labeling the pointed and the barbed ends of actin filaments in cellular networks by permeabilizing cells, following a brief fixation, and introducing labeled actin monomers in the presence or absence of capping protein, respectively. This method quantitatively maps the distributions of free barbed ends and free pointed ends in adherent cells, providing information on the polarity of cytoskeletal structures and mapping active sites available for actin assembly or disassembly. We demonstrate the use of this method by mapping the distribution of actin filament ends in motile fish epithelial keratocytes and in several mammalian cell lines, and show that free barbed ends are enriched near the tip of protruding lamellipodia while free pointed ends concentrate toward the rear.

Original publication

DOI

10.1002/cm.21176

Type

Journal article

Journal

Cytoskeleton (Hoboken, N.J.)

Publication Date

11/06/2014

Volume

71

Pages

341 - 350

Addresses

Department of Physics and the Russell Berrie Nanotechnology Institute, Technion-Israel Institute of Technology, Haifa, Israel.

Keywords

NIH 3T3 Cells, Cytoskeleton, Animals, Fishes, Humans, Mice, Actins, Actin Cytoskeleton