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    Visualization of actin polymerization in invasive structures of macrophages and carcinoma cells using photoconvertible β-actin - Dendra2 fusion proteins

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    Visualization of actin polymer ...
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    Genre
    Journal Article
    Date
    2011-02-28
    Author
    Dovas, A
    Gligorijevic, B
    Chen, X
    Entenberg, D
    Condeelis, J
    Cox, D
    Subject
    Actin Cytoskeleton
    Actins
    Adenocarcinoma
    Animals
    Cell Adhesion
    Cell Tracking
    Cells, Cultured
    Green Fluorescent Proteins
    Macrophages
    Mammary Neoplasms, Animal
    Mice
    Microscopy, Fluorescence
    Neoplasm Invasiveness
    Polymerization
    Protein Multimerization
    Pseudopodia
    Rats
    Recombinant Fusion Proteins
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    Permanent link to this record
    http://hdl.handle.net/20.500.12613/5515
    
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    DOI
    10.1371/journal.pone.0016485
    Abstract
    Actin polymerization controls a range of cellular processes, from intracellular trafficking to cell motility and invasion. Generation and elongation of free barbed ends defines the regions of actively polymerizing actin in cells and, consequently, is of importance in the understanding of the mechanisms through which actin dynamics are regulated. Herein we present a method that does not involve cell permeabilization and provides direct visualization of growing barbed ends using photoswitchable β-actin - Dendra2 constructs expressed in murine macrophage and rat mammary adenocarcinoma cell lines. The method exploits the ability of photoconverted (red) G-actin species to become incorporated into pre-existing (green) actin filaments, visualized in two distinct wavelengths using TIRF microscopy. In growing actin filaments, photoconverted (red) monomers are added to the barbed end while only green monomers are recycled from the pointed end. We demonstrate that incorporation of actin into intact podosomes of macrophages occurs constitutively and is amenable to inhibition by cytochalasin D indicating barbed end incorporation. Additionally, actin polymerization does not occur in quiescent invadopodial precursors of carcinoma cells suggesting that the filaments are capped and following epidermal growth factor stimulation actin incorporation occurs in a single but extended peak. Finally, we show that Dendra2 fused to either the N- or the C-terminus of β-actin profoundly affects its localization and incorporation in distinct F-actin structures in carcinoma cells, thus influencing the ability of monomers to be photoconverted. These data support the use of photoswitchable actin-Dendra2 constructs as powerful tools in the visualization of free barbed ends in living cells. © 2011 Dovas et al.
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    Public Library of Science (PLoS)
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    PLoS ONE
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    http://dx.doi.org/10.34944/dspace/5497
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