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Differential mapping of the free barbed and pointed ends of actin filaments in cells
Author(s) -
Ofer Noa,
Abu Shah Enas,
Keren Kinneret
Publication year - 2014
Publication title -
cytoskeleton
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.95
H-Index - 86
eISSN - 1949-3592
pISSN - 1949-3584
DOI - 10.1002/cm.21176
Subject(s) - lamellipodium , treadmilling , biology , microbiology and biotechnology , actin , cytoskeleton , protein filament , actin cytoskeleton , microfilament , actin remodeling , cell , biochemistry
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. © 2014 Wiley Periodicals, Inc.