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Subcellular targeting of oxidants during endothelial cell migration.

Wu RF, Xu YC, Ma Z, Nwariaku FE, Sarosi GA, Terada LS - J. Cell Biol. (2005)

Bottom Line: Endogenous oxidants participate in endothelial cell migration, suggesting that the enzymatic source of oxidants, like other proteins controlling cell migration, requires precise subcellular localization for spatial confinement of signaling effects.We found that the nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase adaptor p47(phox) and its binding partner TRAF4 were sequestered within nascent, focal complexlike structures in the lamellae of motile endothelial cells.Our data suggest that TRAF4 specifies a molecular address within focal complexes that is targeted for oxidative modification during cell migration.

View Article: PubMed Central - PubMed

Affiliation: University of Texas Southwestern, Dallas, TX 75390, USA.

ABSTRACT
Endogenous oxidants participate in endothelial cell migration, suggesting that the enzymatic source of oxidants, like other proteins controlling cell migration, requires precise subcellular localization for spatial confinement of signaling effects. We found that the nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase adaptor p47(phox) and its binding partner TRAF4 were sequestered within nascent, focal complexlike structures in the lamellae of motile endothelial cells. TRAF4 directly associated with the focal contact scaffold Hic-5, and the knockdown of either protein, disruption of the complex, or oxidant scavenging blocked cell migration. An active mutant of TRAF4 activated the NADPH oxidase downstream of the Rho GTPases and p21-activated kinase 1 (PAK1) and oxidatively modified the focal contact phosphatase PTP-PEST. The oxidase also functioned upstream of Rac1 activation, suggesting its participation in a positive feedback loop. Active TRAF4 initiated robust membrane ruffling through Rac1, PAK1, and the oxidase, whereas the knockdown of PTP-PEST increased ruffling independent of oxidase activation. Our data suggest that TRAF4 specifies a molecular address within focal complexes that is targeted for oxidative modification during cell migration.

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p47phox associates with TRAF4 in focal complexes. (A) Confocal image of DsRed-p47 in small, discrete zyxin-GFP–containing complexes within the leading (right) lamellar edge. Stress fiber–associated focal adhesions (top inset) lack DsRed-p47. DsRed-p47 also localized to the periphery (arrow). (B) TIRF image shows ventral appearance of DsRed-p47 with nascent zyxin-GFP aggregates at the leading edge of a protrusion. (C) Immunofluorescent image of endogenous p47phox (AlexaFluor488) showing colocalization with small vinculin (AlexaFluor555) accumulations. (D) Immunofluorescent image showing the appearance of endogenous TRAF4 (AlexaFluor488) and p47phox (AlexaFluor633) in small dotlike structures at the edge of a protrusion (arrows). B–D, TIRF images. Bars (A and B), 20 μm; (C and D) 10 μm.
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fig2: p47phox associates with TRAF4 in focal complexes. (A) Confocal image of DsRed-p47 in small, discrete zyxin-GFP–containing complexes within the leading (right) lamellar edge. Stress fiber–associated focal adhesions (top inset) lack DsRed-p47. DsRed-p47 also localized to the periphery (arrow). (B) TIRF image shows ventral appearance of DsRed-p47 with nascent zyxin-GFP aggregates at the leading edge of a protrusion. (C) Immunofluorescent image of endogenous p47phox (AlexaFluor488) showing colocalization with small vinculin (AlexaFluor555) accumulations. (D) Immunofluorescent image showing the appearance of endogenous TRAF4 (AlexaFluor488) and p47phox (AlexaFluor633) in small dotlike structures at the edge of a protrusion (arrows). B–D, TIRF images. Bars (A and B), 20 μm; (C and D) 10 μm.

Mentions: The distribution of DsRed-p47 also became highly polarized in migrating endothelial cells, concentrating in zyxin-containing structures within lamellar protrusions (Fig. 2 A). In addition, faint linear accumulation of DsRed-p47 was frequently seen at the outer edge of protrusions before visible organization of zyxin-GFP aggregates. DsRed-p47 was not found within stable focal adhesions in either mobile or static cells. TIRF microscopy also revealed the early appearance of DsRed-p47 at both linear and dotlike ventral structures at the periphery of advancing edges, partially coinciding with zyxin-GFP accumulation (Fig. 2 B). In addition, p47phox specifically coprecipitated with GFP-zyxin (not depicted), which is consistent with its incorporation into focal complex assemblies. By immunofluorescence, endogenous p47phox accurately colocalized with small- to medium-sized vinculin aggregates (Fig. 2 C), and p47phox colocalized with endogenous TRAF4 at the tips of protrusions, which is again consistent with an association within focal complexes (Fig. 2 D).


Subcellular targeting of oxidants during endothelial cell migration.

Wu RF, Xu YC, Ma Z, Nwariaku FE, Sarosi GA, Terada LS - J. Cell Biol. (2005)

p47phox associates with TRAF4 in focal complexes. (A) Confocal image of DsRed-p47 in small, discrete zyxin-GFP–containing complexes within the leading (right) lamellar edge. Stress fiber–associated focal adhesions (top inset) lack DsRed-p47. DsRed-p47 also localized to the periphery (arrow). (B) TIRF image shows ventral appearance of DsRed-p47 with nascent zyxin-GFP aggregates at the leading edge of a protrusion. (C) Immunofluorescent image of endogenous p47phox (AlexaFluor488) showing colocalization with small vinculin (AlexaFluor555) accumulations. (D) Immunofluorescent image showing the appearance of endogenous TRAF4 (AlexaFluor488) and p47phox (AlexaFluor633) in small dotlike structures at the edge of a protrusion (arrows). B–D, TIRF images. Bars (A and B), 20 μm; (C and D) 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171295&req=5

fig2: p47phox associates with TRAF4 in focal complexes. (A) Confocal image of DsRed-p47 in small, discrete zyxin-GFP–containing complexes within the leading (right) lamellar edge. Stress fiber–associated focal adhesions (top inset) lack DsRed-p47. DsRed-p47 also localized to the periphery (arrow). (B) TIRF image shows ventral appearance of DsRed-p47 with nascent zyxin-GFP aggregates at the leading edge of a protrusion. (C) Immunofluorescent image of endogenous p47phox (AlexaFluor488) showing colocalization with small vinculin (AlexaFluor555) accumulations. (D) Immunofluorescent image showing the appearance of endogenous TRAF4 (AlexaFluor488) and p47phox (AlexaFluor633) in small dotlike structures at the edge of a protrusion (arrows). B–D, TIRF images. Bars (A and B), 20 μm; (C and D) 10 μm.
Mentions: The distribution of DsRed-p47 also became highly polarized in migrating endothelial cells, concentrating in zyxin-containing structures within lamellar protrusions (Fig. 2 A). In addition, faint linear accumulation of DsRed-p47 was frequently seen at the outer edge of protrusions before visible organization of zyxin-GFP aggregates. DsRed-p47 was not found within stable focal adhesions in either mobile or static cells. TIRF microscopy also revealed the early appearance of DsRed-p47 at both linear and dotlike ventral structures at the periphery of advancing edges, partially coinciding with zyxin-GFP accumulation (Fig. 2 B). In addition, p47phox specifically coprecipitated with GFP-zyxin (not depicted), which is consistent with its incorporation into focal complex assemblies. By immunofluorescence, endogenous p47phox accurately colocalized with small- to medium-sized vinculin aggregates (Fig. 2 C), and p47phox colocalized with endogenous TRAF4 at the tips of protrusions, which is again consistent with an association within focal complexes (Fig. 2 D).

Bottom Line: Endogenous oxidants participate in endothelial cell migration, suggesting that the enzymatic source of oxidants, like other proteins controlling cell migration, requires precise subcellular localization for spatial confinement of signaling effects.We found that the nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase adaptor p47(phox) and its binding partner TRAF4 were sequestered within nascent, focal complexlike structures in the lamellae of motile endothelial cells.Our data suggest that TRAF4 specifies a molecular address within focal complexes that is targeted for oxidative modification during cell migration.

View Article: PubMed Central - PubMed

Affiliation: University of Texas Southwestern, Dallas, TX 75390, USA.

ABSTRACT
Endogenous oxidants participate in endothelial cell migration, suggesting that the enzymatic source of oxidants, like other proteins controlling cell migration, requires precise subcellular localization for spatial confinement of signaling effects. We found that the nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase adaptor p47(phox) and its binding partner TRAF4 were sequestered within nascent, focal complexlike structures in the lamellae of motile endothelial cells. TRAF4 directly associated with the focal contact scaffold Hic-5, and the knockdown of either protein, disruption of the complex, or oxidant scavenging blocked cell migration. An active mutant of TRAF4 activated the NADPH oxidase downstream of the Rho GTPases and p21-activated kinase 1 (PAK1) and oxidatively modified the focal contact phosphatase PTP-PEST. The oxidase also functioned upstream of Rac1 activation, suggesting its participation in a positive feedback loop. Active TRAF4 initiated robust membrane ruffling through Rac1, PAK1, and the oxidase, whereas the knockdown of PTP-PEST increased ruffling independent of oxidase activation. Our data suggest that TRAF4 specifies a molecular address within focal complexes that is targeted for oxidative modification during cell migration.

Show MeSH
Related in: MedlinePlus