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

Dovas A, Gligorijevic B, Chen X, Entenberg D, Condeelis J, Cox D - PLoS ONE (2011)

Bottom Line: 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.These data support the use of photoswitchable actin-Dendra2 constructs as powerful tools in the visualization of free barbed ends in living cells.

View Article: PubMed Central - PubMed

Affiliation: Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America.

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.

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Photoconversion of β-actin-Dendra2 monomers in MTLn3 cells reveals extended actin polymerization at barbed ends in invadopodial precursors stimulated by EGF.(A,B) Still images of cells that were either left untreated (A) or treated with cytochalasin D (B) taken before the photoconversion (0s), after photoconversion (20s) and after EGF stimulation (240s). Each cell is shown in green, 488nm channel (upper panels) and red, 561nm channel (lower panels). White circle marks the site of photoconversion. Scale bar, 2µm. (C) Representative plots of actin fluorescence of unconverted (488 nm) and photoconverted (561 nm) BAD2 species in an invadopodium of an untreated and a cytochalasin D (cytoD)-treated cell. Arrows point to the time of photoconversion (407 nm) and EGF addition. Cytochalasin D blocks EGF-induced BAD2 incorporation into the invadopodium. (D) Average fluorescence intensity of unconverted (488 nm) and photoconverted (561 nm) BAD2 in invadopodia of cells that were left untreated or treated with cytochalasin D (+cytoD). 0: pre-EGF addition; 1: 1 min after EGF addition; 2: 2 min after EGF addition; 3: 3 min after EGF addition. Values were normalized to pre-photoconversion values and average of intensity in invadopodial precursors from 5 different cells per condition are shown +/−s.e.m.
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pone-0016485-g005: Photoconversion of β-actin-Dendra2 monomers in MTLn3 cells reveals extended actin polymerization at barbed ends in invadopodial precursors stimulated by EGF.(A,B) Still images of cells that were either left untreated (A) or treated with cytochalasin D (B) taken before the photoconversion (0s), after photoconversion (20s) and after EGF stimulation (240s). Each cell is shown in green, 488nm channel (upper panels) and red, 561nm channel (lower panels). White circle marks the site of photoconversion. Scale bar, 2µm. (C) Representative plots of actin fluorescence of unconverted (488 nm) and photoconverted (561 nm) BAD2 species in an invadopodium of an untreated and a cytochalasin D (cytoD)-treated cell. Arrows point to the time of photoconversion (407 nm) and EGF addition. Cytochalasin D blocks EGF-induced BAD2 incorporation into the invadopodium. (D) Average fluorescence intensity of unconverted (488 nm) and photoconverted (561 nm) BAD2 in invadopodia of cells that were left untreated or treated with cytochalasin D (+cytoD). 0: pre-EGF addition; 1: 1 min after EGF addition; 2: 2 min after EGF addition; 3: 3 min after EGF addition. Values were normalized to pre-photoconversion values and average of intensity in invadopodial precursors from 5 different cells per condition are shown +/−s.e.m.

Mentions: We repeated this experiment in MTLn3 cells transfected with BAD2 plated on thin (100 nm) gelatin matrix [16] and serum-starved prior to imaging. A cytoplasmic region with no visible invadopodial precursors was photoconverted and 20-25 seconds later, red BAD2 monomers were homogeneously distributed in the cytoplasm but did not incorporate efficiently into invadopodial precursors (Figure 5A), suggesting that actin filaments in invadopodial precursors of quiescent serum-deprived cells may be capped. Subsequent EGF stimulation resulted in an increase of the intensity in both green (non-converted) and red (photoconverted) BAD2 species at pre-existing invadopodial precursors. However, whereas the green species reached a plateau of intensity at 1 minute post-stimulation, the red species continued to increase, plateauing at 3 minutes (Figure 5 C, D). This indicates that incorporation of actin monomers onto free barbed ends continues up to 3 minutes post-EGF stimulation. Similar to macrophage podosomes, incorporation of photoconverted BAD2 monomers was inhibited in cells that had been pre-treated with cytochalasin D for 1 minute before the photoconversion and EGF stimulation (Figure 5B,C, D) suggesting that incorporation of BAD2 occurred in barbed ends of growing invadopodial precursors. We have been able therefore to measure extended actin incorporation into free barbed ends without the complexity of depolymerization.


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

Dovas A, Gligorijevic B, Chen X, Entenberg D, Condeelis J, Cox D - PLoS ONE (2011)

Photoconversion of β-actin-Dendra2 monomers in MTLn3 cells reveals extended actin polymerization at barbed ends in invadopodial precursors stimulated by EGF.(A,B) Still images of cells that were either left untreated (A) or treated with cytochalasin D (B) taken before the photoconversion (0s), after photoconversion (20s) and after EGF stimulation (240s). Each cell is shown in green, 488nm channel (upper panels) and red, 561nm channel (lower panels). White circle marks the site of photoconversion. Scale bar, 2µm. (C) Representative plots of actin fluorescence of unconverted (488 nm) and photoconverted (561 nm) BAD2 species in an invadopodium of an untreated and a cytochalasin D (cytoD)-treated cell. Arrows point to the time of photoconversion (407 nm) and EGF addition. Cytochalasin D blocks EGF-induced BAD2 incorporation into the invadopodium. (D) Average fluorescence intensity of unconverted (488 nm) and photoconverted (561 nm) BAD2 in invadopodia of cells that were left untreated or treated with cytochalasin D (+cytoD). 0: pre-EGF addition; 1: 1 min after EGF addition; 2: 2 min after EGF addition; 3: 3 min after EGF addition. Values were normalized to pre-photoconversion values and average of intensity in invadopodial precursors from 5 different cells per condition are shown +/−s.e.m.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3038862&req=5

pone-0016485-g005: Photoconversion of β-actin-Dendra2 monomers in MTLn3 cells reveals extended actin polymerization at barbed ends in invadopodial precursors stimulated by EGF.(A,B) Still images of cells that were either left untreated (A) or treated with cytochalasin D (B) taken before the photoconversion (0s), after photoconversion (20s) and after EGF stimulation (240s). Each cell is shown in green, 488nm channel (upper panels) and red, 561nm channel (lower panels). White circle marks the site of photoconversion. Scale bar, 2µm. (C) Representative plots of actin fluorescence of unconverted (488 nm) and photoconverted (561 nm) BAD2 species in an invadopodium of an untreated and a cytochalasin D (cytoD)-treated cell. Arrows point to the time of photoconversion (407 nm) and EGF addition. Cytochalasin D blocks EGF-induced BAD2 incorporation into the invadopodium. (D) Average fluorescence intensity of unconverted (488 nm) and photoconverted (561 nm) BAD2 in invadopodia of cells that were left untreated or treated with cytochalasin D (+cytoD). 0: pre-EGF addition; 1: 1 min after EGF addition; 2: 2 min after EGF addition; 3: 3 min after EGF addition. Values were normalized to pre-photoconversion values and average of intensity in invadopodial precursors from 5 different cells per condition are shown +/−s.e.m.
Mentions: We repeated this experiment in MTLn3 cells transfected with BAD2 plated on thin (100 nm) gelatin matrix [16] and serum-starved prior to imaging. A cytoplasmic region with no visible invadopodial precursors was photoconverted and 20-25 seconds later, red BAD2 monomers were homogeneously distributed in the cytoplasm but did not incorporate efficiently into invadopodial precursors (Figure 5A), suggesting that actin filaments in invadopodial precursors of quiescent serum-deprived cells may be capped. Subsequent EGF stimulation resulted in an increase of the intensity in both green (non-converted) and red (photoconverted) BAD2 species at pre-existing invadopodial precursors. However, whereas the green species reached a plateau of intensity at 1 minute post-stimulation, the red species continued to increase, plateauing at 3 minutes (Figure 5 C, D). This indicates that incorporation of actin monomers onto free barbed ends continues up to 3 minutes post-EGF stimulation. Similar to macrophage podosomes, incorporation of photoconverted BAD2 monomers was inhibited in cells that had been pre-treated with cytochalasin D for 1 minute before the photoconversion and EGF stimulation (Figure 5B,C, D) suggesting that incorporation of BAD2 occurred in barbed ends of growing invadopodial precursors. We have been able therefore to measure extended actin incorporation into free barbed ends without the complexity of depolymerization.

Bottom Line: 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.These data support the use of photoswitchable actin-Dendra2 constructs as powerful tools in the visualization of free barbed ends in living cells.

View Article: PubMed Central - PubMed

Affiliation: Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America.

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.

Show MeSH
Related in: MedlinePlus