Limits...
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.

Show MeSH

Related in: MedlinePlus

TRAF4 accumulates in focal complexes. HUVECs were transfected with the indicated plasmids (color of font indicates pseudocolor designation), plated on fibronectin-coated coverslips, and examined live without fixation using confocal (A and B) or TIRF (C) microscopy. (A) TRAF4-GFP translocated to the tips of leading edges (right), a pattern not seen with pEGFP (left). (B) HUVECs were cotransfected with TRAF4-GFP and DsRed-zyxin. Insets show appearance of TRAF4-GFP and DsRed-zyxin in discontinuous structures at the leading (right) edge of a large protrusion (arrows). Larger stress fiber–anchored focal adhesions contain DsRed-zyxin but no detectable TRAF4-GFP (top insets). (C) TIRF microscopy image of TRAF4-GFP and DsRed-zyxin showing ventral location of small TRAF4-GFP and DsRed-zyxin focal complexes at an advancing protrusion (top inset). Larger DsRed-zyxin focal adhesions lacked TRAF4-GFP (bottom inset). (D) Immunofluorescent images of fixed cells were obtained using TIRF microscopy showing colocalization of endogenous TRAF4 (labeled with AlexaFluor488) within small (closed head arrows) to medium (open head arrow)-sized vinculin aggregates (AlexaFluor555) appearing at the edge of a protrusion. Bars (A–C), 20 μm; (D) 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171295&req=5

fig1: TRAF4 accumulates in focal complexes. HUVECs were transfected with the indicated plasmids (color of font indicates pseudocolor designation), plated on fibronectin-coated coverslips, and examined live without fixation using confocal (A and B) or TIRF (C) microscopy. (A) TRAF4-GFP translocated to the tips of leading edges (right), a pattern not seen with pEGFP (left). (B) HUVECs were cotransfected with TRAF4-GFP and DsRed-zyxin. Insets show appearance of TRAF4-GFP and DsRed-zyxin in discontinuous structures at the leading (right) edge of a large protrusion (arrows). Larger stress fiber–anchored focal adhesions contain DsRed-zyxin but no detectable TRAF4-GFP (top insets). (C) TIRF microscopy image of TRAF4-GFP and DsRed-zyxin showing ventral location of small TRAF4-GFP and DsRed-zyxin focal complexes at an advancing protrusion (top inset). Larger DsRed-zyxin focal adhesions lacked TRAF4-GFP (bottom inset). (D) Immunofluorescent images of fixed cells were obtained using TIRF microscopy showing colocalization of endogenous TRAF4 (labeled with AlexaFluor488) within small (closed head arrows) to medium (open head arrow)-sized vinculin aggregates (AlexaFluor555) appearing at the edge of a protrusion. Bars (A–C), 20 μm; (D) 10 μm.

Mentions: In spontaneously migrating endothelial cells, the distribution of TRAF4-GFP was polarized, concentrating in small discontinuous patches near the lamellipodial edge (Fig. 1 A). Because this pattern resembles that of focal complexes, we examined the colocalization of TRAF4 with zyxin, a scaffolding protein that appears in focal complexes and also accumulates in focal adhesions (Krylyshkina et al., 2003; Franco et al., 2004; Totsukawa et al., 2004). By confocal microscopy, TRAF4-GFP was found to localize within discrete, peripheral DsRed-zyxin aggregates of variable size at the leading edge of endothelial cell lamellar protrusions (Fig. 1 B), along occasional microspike shafts and tips, and discontinuously in a peripheral rim around newly adherent cells, which is consistent with focal complex association (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200507004/DC1). In contrast, TRAF4-GFP accumulation in stable stress fiber–associated focal adhesions was weak or absent (Fig. 1 B). The ventral location of TRAF4-GFP was assessed using total internal reflection fluorescence (TIRF) microscopy, which narrows the z-axis resolution to within 100–150 nm of the coverslip surface. Relatively nonpolarized cells showed accurate colocalization of TRAF4-GFP with peripheral DsRed-zyxin aggregates, confirming targeting to juxtamembrane structures (Fig. S2). In more polarized motile cells, TRAF4-GFP preferentially associated with nascent DsRed-zyxin clusters of variable size at the active leading edge of lamellae, but much less so with larger, rearward focal adhesions (Fig. 1 C). In parallel, immunofluorescent images demonstrated colocalization of endogenous TRAF4 to small (≤1 μm) vinculin-containing structures at the edge of human umbilical vein endothelial cell (HUVEC) protrusions, which is again consistent with its early appearance in focal complexes (Fig. 1 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)

TRAF4 accumulates in focal complexes. HUVECs were transfected with the indicated plasmids (color of font indicates pseudocolor designation), plated on fibronectin-coated coverslips, and examined live without fixation using confocal (A and B) or TIRF (C) microscopy. (A) TRAF4-GFP translocated to the tips of leading edges (right), a pattern not seen with pEGFP (left). (B) HUVECs were cotransfected with TRAF4-GFP and DsRed-zyxin. Insets show appearance of TRAF4-GFP and DsRed-zyxin in discontinuous structures at the leading (right) edge of a large protrusion (arrows). Larger stress fiber–anchored focal adhesions contain DsRed-zyxin but no detectable TRAF4-GFP (top insets). (C) TIRF microscopy image of TRAF4-GFP and DsRed-zyxin showing ventral location of small TRAF4-GFP and DsRed-zyxin focal complexes at an advancing protrusion (top inset). Larger DsRed-zyxin focal adhesions lacked TRAF4-GFP (bottom inset). (D) Immunofluorescent images of fixed cells were obtained using TIRF microscopy showing colocalization of endogenous TRAF4 (labeled with AlexaFluor488) within small (closed head arrows) to medium (open head arrow)-sized vinculin aggregates (AlexaFluor555) appearing at the edge of a protrusion. Bars (A–C), 20 μm; (D) 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171295&req=5

fig1: TRAF4 accumulates in focal complexes. HUVECs were transfected with the indicated plasmids (color of font indicates pseudocolor designation), plated on fibronectin-coated coverslips, and examined live without fixation using confocal (A and B) or TIRF (C) microscopy. (A) TRAF4-GFP translocated to the tips of leading edges (right), a pattern not seen with pEGFP (left). (B) HUVECs were cotransfected with TRAF4-GFP and DsRed-zyxin. Insets show appearance of TRAF4-GFP and DsRed-zyxin in discontinuous structures at the leading (right) edge of a large protrusion (arrows). Larger stress fiber–anchored focal adhesions contain DsRed-zyxin but no detectable TRAF4-GFP (top insets). (C) TIRF microscopy image of TRAF4-GFP and DsRed-zyxin showing ventral location of small TRAF4-GFP and DsRed-zyxin focal complexes at an advancing protrusion (top inset). Larger DsRed-zyxin focal adhesions lacked TRAF4-GFP (bottom inset). (D) Immunofluorescent images of fixed cells were obtained using TIRF microscopy showing colocalization of endogenous TRAF4 (labeled with AlexaFluor488) within small (closed head arrows) to medium (open head arrow)-sized vinculin aggregates (AlexaFluor555) appearing at the edge of a protrusion. Bars (A–C), 20 μm; (D) 10 μm.
Mentions: In spontaneously migrating endothelial cells, the distribution of TRAF4-GFP was polarized, concentrating in small discontinuous patches near the lamellipodial edge (Fig. 1 A). Because this pattern resembles that of focal complexes, we examined the colocalization of TRAF4 with zyxin, a scaffolding protein that appears in focal complexes and also accumulates in focal adhesions (Krylyshkina et al., 2003; Franco et al., 2004; Totsukawa et al., 2004). By confocal microscopy, TRAF4-GFP was found to localize within discrete, peripheral DsRed-zyxin aggregates of variable size at the leading edge of endothelial cell lamellar protrusions (Fig. 1 B), along occasional microspike shafts and tips, and discontinuously in a peripheral rim around newly adherent cells, which is consistent with focal complex association (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200507004/DC1). In contrast, TRAF4-GFP accumulation in stable stress fiber–associated focal adhesions was weak or absent (Fig. 1 B). The ventral location of TRAF4-GFP was assessed using total internal reflection fluorescence (TIRF) microscopy, which narrows the z-axis resolution to within 100–150 nm of the coverslip surface. Relatively nonpolarized cells showed accurate colocalization of TRAF4-GFP with peripheral DsRed-zyxin aggregates, confirming targeting to juxtamembrane structures (Fig. S2). In more polarized motile cells, TRAF4-GFP preferentially associated with nascent DsRed-zyxin clusters of variable size at the active leading edge of lamellae, but much less so with larger, rearward focal adhesions (Fig. 1 C). In parallel, immunofluorescent images demonstrated colocalization of endogenous TRAF4 to small (≤1 μm) vinculin-containing structures at the edge of human umbilical vein endothelial cell (HUVEC) protrusions, which is again consistent with its early appearance in focal complexes (Fig. 1 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