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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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Related in: MedlinePlus

WAVE2 is important for the transcellular route of lymphocyte transmigration and bulk migration of lymphocytes across endothelial monolayers. (A) Representative images illustrating the transcellular route (red) and paracellular route (blue). An endothelial monolayer with transmigrating lymphocytes was stained for ICAM-1, VE-cadherin, and F-actin. (B) Quantification of the routes of transmigration by lymphocytes (PBLs). The percentage of total PBLs that transmigrate by the paracellular or the transcellular route was calculated. Data from experiments on three separate days are combined and plotted as the mean and SE of proportion (control EC monolayers, N = 3330 PBLs; WAVE2-knockdown EC monolayers, N = 2167 PBLs). (C) WAVE2-knockdown endothelial monolayers show increased transendothelial migration by lymphocytes in Transwell assays. Box-and-whisker plots show the median, the interquartile range, and the extremes of the number of transmigrated cells (N = 14).
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Figure 3: WAVE2 is important for the transcellular route of lymphocyte transmigration and bulk migration of lymphocytes across endothelial monolayers. (A) Representative images illustrating the transcellular route (red) and paracellular route (blue). An endothelial monolayer with transmigrating lymphocytes was stained for ICAM-1, VE-cadherin, and F-actin. (B) Quantification of the routes of transmigration by lymphocytes (PBLs). The percentage of total PBLs that transmigrate by the paracellular or the transcellular route was calculated. Data from experiments on three separate days are combined and plotted as the mean and SE of proportion (control EC monolayers, N = 3330 PBLs; WAVE2-knockdown EC monolayers, N = 2167 PBLs). (C) WAVE2-knockdown endothelial monolayers show increased transendothelial migration by lymphocytes in Transwell assays. Box-and-whisker plots show the median, the interquartile range, and the extremes of the number of transmigrated cells (N = 14).

Mentions: To quantitate the effect on WAVE2 depletion on TEM, we first used a conventional Transwell assay with an endothelial monolayer on the filter separating the upper and lower chambers. We measured the number of PBLs that migrated from the upper to the lower chamber. WAVE2 depletion increased the number of PBLs that transmigrated (Figure 3C). However, the level of variation between experiments was large with this assay; in some cases, the number of transmigrating PBLs did not change or decreased. We speculate that this high level of variance in the assay resulted from variations in the morphology of the EC monolayer caused by WAVE2 depletion, which was associated with gaps between ECs of variable number and size. The effect of WAVE2 depletion on EC monolayer morphology is discussed in more detail later. Expression of siRNA-resistant WAVE2 restored PBL transmigration to levels similar to that in control endothelial monolayers (Figure 3C).


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 is important for the transcellular route of lymphocyte transmigration and bulk migration of lymphocytes across endothelial monolayers. (A) Representative images illustrating the transcellular route (red) and paracellular route (blue). An endothelial monolayer with transmigrating lymphocytes was stained for ICAM-1, VE-cadherin, and F-actin. (B) Quantification of the routes of transmigration by lymphocytes (PBLs). The percentage of total PBLs that transmigrate by the paracellular or the transcellular route was calculated. Data from experiments on three separate days are combined and plotted as the mean and SE of proportion (control EC monolayers, N = 3330 PBLs; WAVE2-knockdown EC monolayers, N = 2167 PBLs). (C) WAVE2-knockdown endothelial monolayers show increased transendothelial migration by lymphocytes in Transwell assays. Box-and-whisker plots show the median, the interquartile range, and the extremes of the number of transmigrated cells (N = 14).
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Related In: Results  -  Collection

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Figure 3: WAVE2 is important for the transcellular route of lymphocyte transmigration and bulk migration of lymphocytes across endothelial monolayers. (A) Representative images illustrating the transcellular route (red) and paracellular route (blue). An endothelial monolayer with transmigrating lymphocytes was stained for ICAM-1, VE-cadherin, and F-actin. (B) Quantification of the routes of transmigration by lymphocytes (PBLs). The percentage of total PBLs that transmigrate by the paracellular or the transcellular route was calculated. Data from experiments on three separate days are combined and plotted as the mean and SE of proportion (control EC monolayers, N = 3330 PBLs; WAVE2-knockdown EC monolayers, N = 2167 PBLs). (C) WAVE2-knockdown endothelial monolayers show increased transendothelial migration by lymphocytes in Transwell assays. Box-and-whisker plots show the median, the interquartile range, and the extremes of the number of transmigrated cells (N = 14).
Mentions: To quantitate the effect on WAVE2 depletion on TEM, we first used a conventional Transwell assay with an endothelial monolayer on the filter separating the upper and lower chambers. We measured the number of PBLs that migrated from the upper to the lower chamber. WAVE2 depletion increased the number of PBLs that transmigrated (Figure 3C). However, the level of variation between experiments was large with this assay; in some cases, the number of transmigrating PBLs did not change or decreased. We speculate that this high level of variance in the assay resulted from variations in the morphology of the EC monolayer caused by WAVE2 depletion, which was associated with gaps between ECs of variable number and size. The effect of WAVE2 depletion on EC monolayer morphology is discussed in more detail later. Expression of siRNA-resistant WAVE2 restored PBL transmigration to levels similar to that in control endothelial monolayers (Figure 3C).

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