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

Athanassios Dovas, Bojana Gligorijevic, Xiaoming Chen, David R. Entenberg, John S. Condeelis, Dianne Cox

Research output: Contribution to journalArticle

14 Citations (Scopus)

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.

Original languageEnglish (US)
Article numbere16485
JournalPLoS One
Volume6
Issue number2
DOIs
StatePublished - 2011

Fingerprint

Macrophages
Polymerization
polymerization
carcinoma
actin
Actins
macrophages
Fusion reactions
Visualization
Cells
Carcinoma
Proteins
proteins
cells
microfilaments
Actin Cytoskeleton
Monomers
cytochalasin D
Cytochalasin D
cell invasion

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Visualization of actin polymerization in invasive structures of macrophages and carcinoma cells using photoconvertible β-actin - Dendra2 fusion proteins. / Dovas, Athanassios; Gligorijevic, Bojana; Chen, Xiaoming; Entenberg, David R.; Condeelis, John S.; Cox, Dianne.

In: PLoS One, Vol. 6, No. 2, e16485, 2011.

Research output: Contribution to journalArticle

@article{e443715fe6b54159852d59c685827aed,
title = "Visualization of actin polymerization in invasive structures of macrophages and carcinoma cells using photoconvertible β-actin - Dendra2 fusion proteins",
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.",
author = "Athanassios Dovas and Bojana Gligorijevic and Xiaoming Chen and Entenberg, {David R.} and Condeelis, {John S.} and Dianne Cox",
year = "2011",
doi = "10.1371/journal.pone.0016485",
language = "English (US)",
volume = "6",
journal = "PLoS One",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "2",

}

TY - JOUR

T1 - Visualization of actin polymerization in invasive structures of macrophages and carcinoma cells using photoconvertible β-actin - Dendra2 fusion proteins

AU - Dovas, Athanassios

AU - Gligorijevic, Bojana

AU - Chen, Xiaoming

AU - Entenberg, David R.

AU - Condeelis, John S.

AU - Cox, Dianne

PY - 2011

Y1 - 2011

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=79951926374&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=79951926374&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0016485

DO - 10.1371/journal.pone.0016485

M3 - Article

VL - 6

JO - PLoS One

JF - PLoS One

SN - 1932-6203

IS - 2

M1 - e16485

ER -