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RhoA is required for monocyte tail retraction during transendothelial migration.

Worthylake RA, Lemoine S, Watson JM, Burridge K - J. Cell Biol. (2001)

Bottom Line: We have analyzed the function of RhoA in the cytoskeletal reorganizations that occur during transmigration.We also demonstrate that p160ROCK, a serine/threonine kinase effector of RhoA, is both necessary and sufficient for RhoA-mediated tail retraction.Finally, we find that p160ROCK signaling negatively regulates integrin adhesions and that inhibition of RhoA results in an accumulation of beta2 integrin in the unretracted tails.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. becky_worthylake@med.unc.edu

ABSTRACT
Transendothelial migration of monocytes is the process by which monocytes leave the circulatory system and extravasate through the endothelial lining of the blood vessel wall and enter the underlying tissue. Transmigration requires coordination of alterations in cell shape and adhesive properties that are mediated by cytoskeletal dynamics. We have analyzed the function of RhoA in the cytoskeletal reorganizations that occur during transmigration. By loading monocytes with C3, an inhibitor of RhoA, we found that RhoA was required for transendothelial migration. We then examined individual steps of transmigration to explore the requirement for RhoA in extravasation. Our studies showed that RhoA was not required for monocyte attachment to the endothelium nor subsequent spreading of the monocyte on the endothelial surface. Time-lapse video microscopy analysis revealed that C3-loaded monocytes also had significant forward crawling movement on the endothelial monolayer and were able to invade between neighboring endothelial cells. However, RhoA was required to retract the tail of the migrating monocyte and complete diapedesis. We also demonstrate that p160ROCK, a serine/threonine kinase effector of RhoA, is both necessary and sufficient for RhoA-mediated tail retraction. Finally, we find that p160ROCK signaling negatively regulates integrin adhesions and that inhibition of RhoA results in an accumulation of beta2 integrin in the unretracted tails.

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RhoA is not required for adhesion to the endothelium. Fluorescently labeled GST- (control) or C3-loaded monocytes were plated onto resting or IL-1–stimulated endothelial monolayers grown in 96 well plates. After 15 min, the cocultures were washed with PBS, and adhesion of the monocytes was quantitated by measuring the fluorescence. Data plotted are the average of triplicate wells from a typical experiment.
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fig2: RhoA is not required for adhesion to the endothelium. Fluorescently labeled GST- (control) or C3-loaded monocytes were plated onto resting or IL-1–stimulated endothelial monolayers grown in 96 well plates. After 15 min, the cocultures were washed with PBS, and adhesion of the monocytes was quantitated by measuring the fluorescence. Data plotted are the average of triplicate wells from a typical experiment.

Mentions: Transmigration was divided into three distinct steps that could be measured experimentally. First, cells adhere to the endothelium through a wide range of molecular interactions including glycoproteins, Ig-containing adhesion molecules, and integrins. Second, leukocytes spread into a more flattened morphology and extend membrane to migrate along and invade between neighboring endothelial cells. Finally, cells complete diapedesis through the endothelial monolayer to reach the underlying tissue. Thus, we asked which step of transmigration required RhoA activity. To measure the initial attachment of monocytes to endothelial monolayers, the number of monocytes per field that were adherent at 15 min, were counted by microscopy. Because the surface expression of many endothelial adhesion molecules requires activation of inflammatory cytokines, we compared the adhesion of monocytes to resting or activated monolayers. Fig. 2 shows that activation of endothelial cells with IL-1 increased the adhesion of monocytes ∼10-fold. We found that inhibition of RhoA by C3 loading had no effect on the ability of monocytes to adhere to either resting or activated endothelium.


RhoA is required for monocyte tail retraction during transendothelial migration.

Worthylake RA, Lemoine S, Watson JM, Burridge K - J. Cell Biol. (2001)

RhoA is not required for adhesion to the endothelium. Fluorescently labeled GST- (control) or C3-loaded monocytes were plated onto resting or IL-1–stimulated endothelial monolayers grown in 96 well plates. After 15 min, the cocultures were washed with PBS, and adhesion of the monocytes was quantitated by measuring the fluorescence. Data plotted are the average of triplicate wells from a typical experiment.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196864&req=5

fig2: RhoA is not required for adhesion to the endothelium. Fluorescently labeled GST- (control) or C3-loaded monocytes were plated onto resting or IL-1–stimulated endothelial monolayers grown in 96 well plates. After 15 min, the cocultures were washed with PBS, and adhesion of the monocytes was quantitated by measuring the fluorescence. Data plotted are the average of triplicate wells from a typical experiment.
Mentions: Transmigration was divided into three distinct steps that could be measured experimentally. First, cells adhere to the endothelium through a wide range of molecular interactions including glycoproteins, Ig-containing adhesion molecules, and integrins. Second, leukocytes spread into a more flattened morphology and extend membrane to migrate along and invade between neighboring endothelial cells. Finally, cells complete diapedesis through the endothelial monolayer to reach the underlying tissue. Thus, we asked which step of transmigration required RhoA activity. To measure the initial attachment of monocytes to endothelial monolayers, the number of monocytes per field that were adherent at 15 min, were counted by microscopy. Because the surface expression of many endothelial adhesion molecules requires activation of inflammatory cytokines, we compared the adhesion of monocytes to resting or activated monolayers. Fig. 2 shows that activation of endothelial cells with IL-1 increased the adhesion of monocytes ∼10-fold. We found that inhibition of RhoA by C3 loading had no effect on the ability of monocytes to adhere to either resting or activated endothelium.

Bottom Line: We have analyzed the function of RhoA in the cytoskeletal reorganizations that occur during transmigration.We also demonstrate that p160ROCK, a serine/threonine kinase effector of RhoA, is both necessary and sufficient for RhoA-mediated tail retraction.Finally, we find that p160ROCK signaling negatively regulates integrin adhesions and that inhibition of RhoA results in an accumulation of beta2 integrin in the unretracted tails.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. becky_worthylake@med.unc.edu

ABSTRACT
Transendothelial migration of monocytes is the process by which monocytes leave the circulatory system and extravasate through the endothelial lining of the blood vessel wall and enter the underlying tissue. Transmigration requires coordination of alterations in cell shape and adhesive properties that are mediated by cytoskeletal dynamics. We have analyzed the function of RhoA in the cytoskeletal reorganizations that occur during transmigration. By loading monocytes with C3, an inhibitor of RhoA, we found that RhoA was required for transendothelial migration. We then examined individual steps of transmigration to explore the requirement for RhoA in extravasation. Our studies showed that RhoA was not required for monocyte attachment to the endothelium nor subsequent spreading of the monocyte on the endothelial surface. Time-lapse video microscopy analysis revealed that C3-loaded monocytes also had significant forward crawling movement on the endothelial monolayer and were able to invade between neighboring endothelial cells. However, RhoA was required to retract the tail of the migrating monocyte and complete diapedesis. We also demonstrate that p160ROCK, a serine/threonine kinase effector of RhoA, is both necessary and sufficient for RhoA-mediated tail retraction. Finally, we find that p160ROCK signaling negatively regulates integrin adhesions and that inhibition of RhoA results in an accumulation of beta2 integrin in the unretracted tails.

Show MeSH
Related in: MedlinePlus