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Endothelial cells use dynamic actin to facilitate lymphocyte transendothelial migration and maintain the monolayer barrier.

Mooren OL, Li J, Nawas J, Cooper JA - Mol. Biol. Cell (2014)

Bottom Line: The actin cytoskeleton of the endothelial cell (EC) is known to facilitate transmigration, but the cellular and molecular mechanisms are not well understood.We found that docking structure formation involves the localization and activation of Arp2/3 complex by WAVE2.Finally, we found that ECs in resting endothelial monolayers use lamellipodial protrusions dependent on WAVE2 to form and maintain contacts and junctions between cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University, St. Louis, MO 63110.

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WAVE2 maintains endothelial monolayer integrity by promoting Arp2/3-based lamellipodial protrusions. (A) Electrical resistance across monolayers, plotted as a normalized index for TER (see Materials and Methods). Data are derived from experiments on three or more days, with three or more repetitions on each day. Plotted values are the mean of all the data points, and error bars are SD (N ≥ 9). (B) Localization of WAVE2 and Arp2/3 complex adjacent to cell–cell junctions, based on immunofluorescence and fluorescent phalloidin staining of endothelial monolayers. Arrows indicate localization close to but not coincident with cell junctions. Scale bar, 50 μm. (C) Magnified views of boxed regions in B. Line scans across cell–cell junctions (blue box) or along junctions (magenta box). Scale bar, 20 μm. (D) Localization of Arp2/3 complex and WAVE2 at cell–cell junctions formed in response to S1P. Merge, green is anti-Arp3 or anti-WAVE2; red is VE-cadherin. Top, control; bottom, with S1P added. Scale bar, 50 μm; inset, 20 μm.
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Figure 5: WAVE2 maintains endothelial monolayer integrity by promoting Arp2/3-based lamellipodial protrusions. (A) Electrical resistance across monolayers, plotted as a normalized index for TER (see Materials and Methods). Data are derived from experiments on three or more days, with three or more repetitions on each day. Plotted values are the mean of all the data points, and error bars are SD (N ≥ 9). (B) Localization of WAVE2 and Arp2/3 complex adjacent to cell–cell junctions, based on immunofluorescence and fluorescent phalloidin staining of endothelial monolayers. Arrows indicate localization close to but not coincident with cell junctions. Scale bar, 50 μm. (C) Magnified views of boxed regions in B. Line scans across cell–cell junctions (blue box) or along junctions (magenta box). Scale bar, 20 μm. (D) Localization of Arp2/3 complex and WAVE2 at cell–cell junctions formed in response to S1P. Merge, green is anti-Arp3 or anti-WAVE2; red is VE-cadherin. Top, control; bottom, with S1P added. Scale bar, 50 μm; inset, 20 μm.

Mentions: On the basis of these observations of EC monolayers, we hypothesized that WAVE2 depletion might cause defects in the overall integrity of the endothelial barrier measured by conventional physiological assays. To test this hypothesis, we first measured electrical resistance across the monolayer (transendothelial resistance [TER]; Figure 5A). WAVE2 depletion caused TER to decrease to levels similar to those caused by chelation of Ca2+, which completely disrupts cadherin-based cell–cell junctions. Depolymerizing actin with latrunculin A (LatA) also decreased TER by a large amount (Figure 5A) and created large gaps between ECs (Supplemental Figure S1B), consistent with previous findings (Prasain and Stevens, 2009). Second, we measured the permeability of the monolayer to fluorescent dextran, as an indicator of barrier integrity. Again, EC monolayers depleted of WAVE2 showed increased permeability compared with control (Supplemental Figure S1C).


Endothelial cells use dynamic actin to facilitate lymphocyte transendothelial migration and maintain the monolayer barrier.

Mooren OL, Li J, Nawas J, Cooper JA - Mol. Biol. Cell (2014)

WAVE2 maintains endothelial monolayer integrity by promoting Arp2/3-based lamellipodial protrusions. (A) Electrical resistance across monolayers, plotted as a normalized index for TER (see Materials and Methods). Data are derived from experiments on three or more days, with three or more repetitions on each day. Plotted values are the mean of all the data points, and error bars are SD (N ≥ 9). (B) Localization of WAVE2 and Arp2/3 complex adjacent to cell–cell junctions, based on immunofluorescence and fluorescent phalloidin staining of endothelial monolayers. Arrows indicate localization close to but not coincident with cell junctions. Scale bar, 50 μm. (C) Magnified views of boxed regions in B. Line scans across cell–cell junctions (blue box) or along junctions (magenta box). Scale bar, 20 μm. (D) Localization of Arp2/3 complex and WAVE2 at cell–cell junctions formed in response to S1P. Merge, green is anti-Arp3 or anti-WAVE2; red is VE-cadherin. Top, control; bottom, with S1P added. Scale bar, 50 μm; inset, 20 μm.
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Figure 5: WAVE2 maintains endothelial monolayer integrity by promoting Arp2/3-based lamellipodial protrusions. (A) Electrical resistance across monolayers, plotted as a normalized index for TER (see Materials and Methods). Data are derived from experiments on three or more days, with three or more repetitions on each day. Plotted values are the mean of all the data points, and error bars are SD (N ≥ 9). (B) Localization of WAVE2 and Arp2/3 complex adjacent to cell–cell junctions, based on immunofluorescence and fluorescent phalloidin staining of endothelial monolayers. Arrows indicate localization close to but not coincident with cell junctions. Scale bar, 50 μm. (C) Magnified views of boxed regions in B. Line scans across cell–cell junctions (blue box) or along junctions (magenta box). Scale bar, 20 μm. (D) Localization of Arp2/3 complex and WAVE2 at cell–cell junctions formed in response to S1P. Merge, green is anti-Arp3 or anti-WAVE2; red is VE-cadherin. Top, control; bottom, with S1P added. Scale bar, 50 μm; inset, 20 μm.
Mentions: On the basis of these observations of EC monolayers, we hypothesized that WAVE2 depletion might cause defects in the overall integrity of the endothelial barrier measured by conventional physiological assays. To test this hypothesis, we first measured electrical resistance across the monolayer (transendothelial resistance [TER]; Figure 5A). WAVE2 depletion caused TER to decrease to levels similar to those caused by chelation of Ca2+, which completely disrupts cadherin-based cell–cell junctions. Depolymerizing actin with latrunculin A (LatA) also decreased TER by a large amount (Figure 5A) and created large gaps between ECs (Supplemental Figure S1B), consistent with previous findings (Prasain and Stevens, 2009). Second, we measured the permeability of the monolayer to fluorescent dextran, as an indicator of barrier integrity. Again, EC monolayers depleted of WAVE2 showed increased permeability compared with control (Supplemental Figure S1C).

Bottom Line: The actin cytoskeleton of the endothelial cell (EC) is known to facilitate transmigration, but the cellular and molecular mechanisms are not well understood.We found that docking structure formation involves the localization and activation of Arp2/3 complex by WAVE2.Finally, we found that ECs in resting endothelial monolayers use lamellipodial protrusions dependent on WAVE2 to form and maintain contacts and junctions between cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University, St. Louis, MO 63110.

Show MeSH
Related in: MedlinePlus