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Involvement of local lamellipodia in endothelial barrier function.

Breslin JW, Zhang XE, Worthylake RA, Souza-Smith FM - PLoS ONE (2015)

Bottom Line: Blebbistatin also significantly decreased TER of cultured endothelial cells and increased permeability of isolated rat mesenteric venules.Overexpression of Rac1 elevated, while NSC23766 and dominant negative Rac1 reduced barrier function and lamellipodia activity.Combined, these data suggest that local lamellipodia, driven by myosin II and Rac1, are important for dynamic changes in endothelial barrier integrity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, Florida, United States of America.

ABSTRACT
Recently we observed that endothelial cells cultured in tightly confluent monolayers display frequent local lamellipodia, and that thrombin, an agent that increases endothelial permeability, reduces lamellipodia protrusions. This led us to test the hypothesis that local lamellipodia contribute to endothelial barrier function. Movements of subcellular structures containing GFP-actin or VE-cadherin-GFP expressed in endothelial cells were recorded using time-lapse microscopy. Transendothelial electrical resistance (TER) served as an index of endothelial barrier function. Changes in both lamellipodia dynamics and TER were assessed during baseline and after cells were treated with either the barrier-disrupting agent thrombin, or the barrier-stabilizing agent sphingosine-1-phosphate (S1P). The myosin II inhibitor blebbistatin was used to selectively block lamellipodia formation, and was used to test their role in the barrier function of endothelial cell monolayers and isolated, perfused rat mesenteric venules. Myosin light chain (MLC) phosphorylation was assessed by immunofluorescence microscopy. Rac1 and RhoA activation were evaluated using G-LISA assays. The role of Rac1 was tested with the specific inhibitor NSC23766 or by expressing wild-type or dominant negative GFP-Rac1. The results show that thrombin rapidly decreased both TER and the lamellipodia protrusion frequency. S1P rapidly increased TER in association with increased protrusion frequency. Blebbistatin nearly abolished local lamellipodia protrusions while cortical actin fibers and stress fibers remained intact. Blebbistatin also significantly decreased TER of cultured endothelial cells and increased permeability of isolated rat mesenteric venules. Both thrombin and S1P increased MLC phosphorylation and activation of RhoA. However, thrombin and S1P had differential impacts on Rac1, correlating with the changes in TER and lamellipodia protrusion frequency. Overexpression of Rac1 elevated, while NSC23766 and dominant negative Rac1 reduced barrier function and lamellipodia activity. Combined, these data suggest that local lamellipodia, driven by myosin II and Rac1, are important for dynamic changes in endothelial barrier integrity.

No MeSH data available.


Related in: MedlinePlus

Local lamellipodia protruded beyond endothelial adherens junctions containing VE-cadherin-GFP and were associated with junction stability.At the top of all three panels, an image of HUVEC expressing VE-cadherin-GFP is shown. The bounding box in each top image shows the area studied in the time-lapse montages below. Confluent monolayers were used for all experiments, but not all cells expressed detectable levels of VE-cadherin-GFP. A. Time-lapse imaging revealed that VE-cadherin-GFP was most intense at intercellular junctions and in vesicles around the nucleus. Select time-lapse images (from S6 Movie) of the area in the box from top panel show the protrusion and withdrawal of a local lamellipodium (arrows) that spread toward the cell in the top of the image from the belt of VE-cadherin-GFP located between two cells. B. The same cells were tracked just before and during 1 U/ml thrombin treatment. Selected time-lapse images from the bounding box in the top panel (from S7 Movie) show how the withdrawal of a local lamellipodium that had protruded prior to thrombin treatment yielded filopodia-like structures containing VE-cadherin (arrows). Subsequently, as fewer lamellipodia protruded from the cell edge, breaks in the continuous belt of VE-cadherin emerged (arrowheads). C. Time-lapse studies before and after treatment with 2 μM S1P (from S8 Movie) show that lamellipodia spread beyond the VE-cadherin-GFP-rich junctions (arrows). In addition, over time the junctional areas containing VE-cadherin-GFP appeared wider than during baseline (compare the calipers at BL and 20 min). Images are representative of observations from at least three different experiments each with thrombin and S1P.
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pone.0117970.g004: Local lamellipodia protruded beyond endothelial adherens junctions containing VE-cadherin-GFP and were associated with junction stability.At the top of all three panels, an image of HUVEC expressing VE-cadherin-GFP is shown. The bounding box in each top image shows the area studied in the time-lapse montages below. Confluent monolayers were used for all experiments, but not all cells expressed detectable levels of VE-cadherin-GFP. A. Time-lapse imaging revealed that VE-cadherin-GFP was most intense at intercellular junctions and in vesicles around the nucleus. Select time-lapse images (from S6 Movie) of the area in the box from top panel show the protrusion and withdrawal of a local lamellipodium (arrows) that spread toward the cell in the top of the image from the belt of VE-cadherin-GFP located between two cells. B. The same cells were tracked just before and during 1 U/ml thrombin treatment. Selected time-lapse images from the bounding box in the top panel (from S7 Movie) show how the withdrawal of a local lamellipodium that had protruded prior to thrombin treatment yielded filopodia-like structures containing VE-cadherin (arrows). Subsequently, as fewer lamellipodia protruded from the cell edge, breaks in the continuous belt of VE-cadherin emerged (arrowheads). C. Time-lapse studies before and after treatment with 2 μM S1P (from S8 Movie) show that lamellipodia spread beyond the VE-cadherin-GFP-rich junctions (arrows). In addition, over time the junctional areas containing VE-cadherin-GFP appeared wider than during baseline (compare the calipers at BL and 20 min). Images are representative of observations from at least three different experiments each with thrombin and S1P.

Mentions: Because endothelial cell-cell junctions have a key role in regulating paracellular transport [1,45], we investigated the dynamics of the endothelial junction protein VE-cadherin using confluent HUVEC expressing a VE-cadherin-GFP fusion protein. VE-cadherin-GFP was detected predominantly at the cell periphery in a similar fashion as previously reported for native VE-cadherin by immunofluorescent labeling [46,47], and also was very intense in vesicles around the nucleus (S6 Movie). In addition, enough of the expressed VE-cadherin-GFP was located ubiquitously to detect an entire cell’s footprint. This enabled observation of numerous lamellipodia protruding beyond the belt of VE-cadherin-GFP located at intercellular junctions, causing transient overlapping with adjacent cells (Fig. 4A). Most lamellipodia did not appear to have high amounts of VE-cadherin-GFP, although occasionally there were exceptions, such as the newly forming lamellipodium in the last image of the montage in Fig. 4A. Compared to local lamellipodia, most of the belt of VE-cadherin-GFP at endothelial cell-cell junctions was very stable and its movements were relatively slow.


Involvement of local lamellipodia in endothelial barrier function.

Breslin JW, Zhang XE, Worthylake RA, Souza-Smith FM - PLoS ONE (2015)

Local lamellipodia protruded beyond endothelial adherens junctions containing VE-cadherin-GFP and were associated with junction stability.At the top of all three panels, an image of HUVEC expressing VE-cadherin-GFP is shown. The bounding box in each top image shows the area studied in the time-lapse montages below. Confluent monolayers were used for all experiments, but not all cells expressed detectable levels of VE-cadherin-GFP. A. Time-lapse imaging revealed that VE-cadherin-GFP was most intense at intercellular junctions and in vesicles around the nucleus. Select time-lapse images (from S6 Movie) of the area in the box from top panel show the protrusion and withdrawal of a local lamellipodium (arrows) that spread toward the cell in the top of the image from the belt of VE-cadherin-GFP located between two cells. B. The same cells were tracked just before and during 1 U/ml thrombin treatment. Selected time-lapse images from the bounding box in the top panel (from S7 Movie) show how the withdrawal of a local lamellipodium that had protruded prior to thrombin treatment yielded filopodia-like structures containing VE-cadherin (arrows). Subsequently, as fewer lamellipodia protruded from the cell edge, breaks in the continuous belt of VE-cadherin emerged (arrowheads). C. Time-lapse studies before and after treatment with 2 μM S1P (from S8 Movie) show that lamellipodia spread beyond the VE-cadherin-GFP-rich junctions (arrows). In addition, over time the junctional areas containing VE-cadherin-GFP appeared wider than during baseline (compare the calipers at BL and 20 min). Images are representative of observations from at least three different experiments each with thrombin and S1P.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4320108&req=5

pone.0117970.g004: Local lamellipodia protruded beyond endothelial adherens junctions containing VE-cadherin-GFP and were associated with junction stability.At the top of all three panels, an image of HUVEC expressing VE-cadherin-GFP is shown. The bounding box in each top image shows the area studied in the time-lapse montages below. Confluent monolayers were used for all experiments, but not all cells expressed detectable levels of VE-cadherin-GFP. A. Time-lapse imaging revealed that VE-cadherin-GFP was most intense at intercellular junctions and in vesicles around the nucleus. Select time-lapse images (from S6 Movie) of the area in the box from top panel show the protrusion and withdrawal of a local lamellipodium (arrows) that spread toward the cell in the top of the image from the belt of VE-cadherin-GFP located between two cells. B. The same cells were tracked just before and during 1 U/ml thrombin treatment. Selected time-lapse images from the bounding box in the top panel (from S7 Movie) show how the withdrawal of a local lamellipodium that had protruded prior to thrombin treatment yielded filopodia-like structures containing VE-cadherin (arrows). Subsequently, as fewer lamellipodia protruded from the cell edge, breaks in the continuous belt of VE-cadherin emerged (arrowheads). C. Time-lapse studies before and after treatment with 2 μM S1P (from S8 Movie) show that lamellipodia spread beyond the VE-cadherin-GFP-rich junctions (arrows). In addition, over time the junctional areas containing VE-cadherin-GFP appeared wider than during baseline (compare the calipers at BL and 20 min). Images are representative of observations from at least three different experiments each with thrombin and S1P.
Mentions: Because endothelial cell-cell junctions have a key role in regulating paracellular transport [1,45], we investigated the dynamics of the endothelial junction protein VE-cadherin using confluent HUVEC expressing a VE-cadherin-GFP fusion protein. VE-cadherin-GFP was detected predominantly at the cell periphery in a similar fashion as previously reported for native VE-cadherin by immunofluorescent labeling [46,47], and also was very intense in vesicles around the nucleus (S6 Movie). In addition, enough of the expressed VE-cadherin-GFP was located ubiquitously to detect an entire cell’s footprint. This enabled observation of numerous lamellipodia protruding beyond the belt of VE-cadherin-GFP located at intercellular junctions, causing transient overlapping with adjacent cells (Fig. 4A). Most lamellipodia did not appear to have high amounts of VE-cadherin-GFP, although occasionally there were exceptions, such as the newly forming lamellipodium in the last image of the montage in Fig. 4A. Compared to local lamellipodia, most of the belt of VE-cadherin-GFP at endothelial cell-cell junctions was very stable and its movements were relatively slow.

Bottom Line: Blebbistatin also significantly decreased TER of cultured endothelial cells and increased permeability of isolated rat mesenteric venules.Overexpression of Rac1 elevated, while NSC23766 and dominant negative Rac1 reduced barrier function and lamellipodia activity.Combined, these data suggest that local lamellipodia, driven by myosin II and Rac1, are important for dynamic changes in endothelial barrier integrity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, Florida, United States of America.

ABSTRACT
Recently we observed that endothelial cells cultured in tightly confluent monolayers display frequent local lamellipodia, and that thrombin, an agent that increases endothelial permeability, reduces lamellipodia protrusions. This led us to test the hypothesis that local lamellipodia contribute to endothelial barrier function. Movements of subcellular structures containing GFP-actin or VE-cadherin-GFP expressed in endothelial cells were recorded using time-lapse microscopy. Transendothelial electrical resistance (TER) served as an index of endothelial barrier function. Changes in both lamellipodia dynamics and TER were assessed during baseline and after cells were treated with either the barrier-disrupting agent thrombin, or the barrier-stabilizing agent sphingosine-1-phosphate (S1P). The myosin II inhibitor blebbistatin was used to selectively block lamellipodia formation, and was used to test their role in the barrier function of endothelial cell monolayers and isolated, perfused rat mesenteric venules. Myosin light chain (MLC) phosphorylation was assessed by immunofluorescence microscopy. Rac1 and RhoA activation were evaluated using G-LISA assays. The role of Rac1 was tested with the specific inhibitor NSC23766 or by expressing wild-type or dominant negative GFP-Rac1. The results show that thrombin rapidly decreased both TER and the lamellipodia protrusion frequency. S1P rapidly increased TER in association with increased protrusion frequency. Blebbistatin nearly abolished local lamellipodia protrusions while cortical actin fibers and stress fibers remained intact. Blebbistatin also significantly decreased TER of cultured endothelial cells and increased permeability of isolated rat mesenteric venules. Both thrombin and S1P increased MLC phosphorylation and activation of RhoA. However, thrombin and S1P had differential impacts on Rac1, correlating with the changes in TER and lamellipodia protrusion frequency. Overexpression of Rac1 elevated, while NSC23766 and dominant negative Rac1 reduced barrier function and lamellipodia activity. Combined, these data suggest that local lamellipodia, driven by myosin II and Rac1, are important for dynamic changes in endothelial barrier integrity.

No MeSH data available.


Related in: MedlinePlus