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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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p160ROCK is both necessary and sufficient for RhoA-mediated tail retraction. (A) Monocytes were plated onto coverslips in the presence of 10% autologous serum with or without the p160ROCK inhibitor, Y-27632 (10 μM). The graph represents data obtained from monocytes plated onto coverslips with serum containing media and a 1.0–10 μM range of Y-27632 for 45 min. Cells were then fixed, stained for F-actin, and the percentage of cells with tails was scored. The data plotted represent the average from three separate experiments. After 45 min incubation, cells were fixed and stained for F-actin to reveal cell morphology. (B) Monocytes were electroporated with C3 + CA ROCK and compared with those electroporated with C3 + GST. Cells were plated on coverslips with serum-containing media for 45 min before fixation. Cells were stained for F-actin for morphological assessment. (C) Fluorescently labeled monocytes were pretreated with 10 μM Y-27632 for 15 min and then washed and added to activated endothelial monolayers in the presence of 1 μM Y-27632 for 20 min before fixation. Images reveal the morphology of only the monocytes in the coculture. Cells on the left of each pair of images were on top of the endothelial monolayer, as judged by the focal plane. Cells on the right were underneath the monolayer (controls), or caught between neighboring endothelial cells (Y-27632). Bars, 20 μm.
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fig6: p160ROCK is both necessary and sufficient for RhoA-mediated tail retraction. (A) Monocytes were plated onto coverslips in the presence of 10% autologous serum with or without the p160ROCK inhibitor, Y-27632 (10 μM). The graph represents data obtained from monocytes plated onto coverslips with serum containing media and a 1.0–10 μM range of Y-27632 for 45 min. Cells were then fixed, stained for F-actin, and the percentage of cells with tails was scored. The data plotted represent the average from three separate experiments. After 45 min incubation, cells were fixed and stained for F-actin to reveal cell morphology. (B) Monocytes were electroporated with C3 + CA ROCK and compared with those electroporated with C3 + GST. Cells were plated on coverslips with serum-containing media for 45 min before fixation. Cells were stained for F-actin for morphological assessment. (C) Fluorescently labeled monocytes were pretreated with 10 μM Y-27632 for 15 min and then washed and added to activated endothelial monolayers in the presence of 1 μM Y-27632 for 20 min before fixation. Images reveal the morphology of only the monocytes in the coculture. Cells on the left of each pair of images were on top of the endothelial monolayer, as judged by the focal plane. Cells on the right were underneath the monolayer (controls), or caught between neighboring endothelial cells (Y-27632). Bars, 20 μm.

Mentions: To further investigate the signal transduction pathway that leads to tail retraction, we used a specific inhibitor of p160ROCK, Y-27632 (Uehata et al., 1997). This pharmacological reagent is reported to be specific for p160ROCK at 10 μM (Ishizaki et al., 2000), although the possibility exists that Y-27632 may have additional targets. p160ROCK is a serine/threonine kinase that is activated by RhoA and has been shown to be a critical modulator of RhoA-mediated actin dynamics (Amano et al., 2000). To examine the role of p160ROCK in monocyte tail retraction, we plated primary monocytes on coverslips in the presence of 10 μM of Y-27632 and then stained for F-actin to visualize cell morphology. Cells treated with the p160ROCK inhibitor showed morphological abnormalities similar to those observed in C3-loaded monocytes, albeit less severe (Fig. 6 A). The percentage of cells with tails was quantitated over a range of Y-27632 concentrations. We found that even at 1 μM, the percentage of cells with detectable tails nearly doubled over controls. Both the percentage of cells with tails, and the length of the unretracted tails increased with the concentration of Y-27632 (Fig. 6 A).


RhoA is required for monocyte tail retraction during transendothelial migration.

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

p160ROCK is both necessary and sufficient for RhoA-mediated tail retraction. (A) Monocytes were plated onto coverslips in the presence of 10% autologous serum with or without the p160ROCK inhibitor, Y-27632 (10 μM). The graph represents data obtained from monocytes plated onto coverslips with serum containing media and a 1.0–10 μM range of Y-27632 for 45 min. Cells were then fixed, stained for F-actin, and the percentage of cells with tails was scored. The data plotted represent the average from three separate experiments. After 45 min incubation, cells were fixed and stained for F-actin to reveal cell morphology. (B) Monocytes were electroporated with C3 + CA ROCK and compared with those electroporated with C3 + GST. Cells were plated on coverslips with serum-containing media for 45 min before fixation. Cells were stained for F-actin for morphological assessment. (C) Fluorescently labeled monocytes were pretreated with 10 μM Y-27632 for 15 min and then washed and added to activated endothelial monolayers in the presence of 1 μM Y-27632 for 20 min before fixation. Images reveal the morphology of only the monocytes in the coculture. Cells on the left of each pair of images were on top of the endothelial monolayer, as judged by the focal plane. Cells on the right were underneath the monolayer (controls), or caught between neighboring endothelial cells (Y-27632). Bars, 20 μm.
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fig6: p160ROCK is both necessary and sufficient for RhoA-mediated tail retraction. (A) Monocytes were plated onto coverslips in the presence of 10% autologous serum with or without the p160ROCK inhibitor, Y-27632 (10 μM). The graph represents data obtained from monocytes plated onto coverslips with serum containing media and a 1.0–10 μM range of Y-27632 for 45 min. Cells were then fixed, stained for F-actin, and the percentage of cells with tails was scored. The data plotted represent the average from three separate experiments. After 45 min incubation, cells were fixed and stained for F-actin to reveal cell morphology. (B) Monocytes were electroporated with C3 + CA ROCK and compared with those electroporated with C3 + GST. Cells were plated on coverslips with serum-containing media for 45 min before fixation. Cells were stained for F-actin for morphological assessment. (C) Fluorescently labeled monocytes were pretreated with 10 μM Y-27632 for 15 min and then washed and added to activated endothelial monolayers in the presence of 1 μM Y-27632 for 20 min before fixation. Images reveal the morphology of only the monocytes in the coculture. Cells on the left of each pair of images were on top of the endothelial monolayer, as judged by the focal plane. Cells on the right were underneath the monolayer (controls), or caught between neighboring endothelial cells (Y-27632). Bars, 20 μm.
Mentions: To further investigate the signal transduction pathway that leads to tail retraction, we used a specific inhibitor of p160ROCK, Y-27632 (Uehata et al., 1997). This pharmacological reagent is reported to be specific for p160ROCK at 10 μM (Ishizaki et al., 2000), although the possibility exists that Y-27632 may have additional targets. p160ROCK is a serine/threonine kinase that is activated by RhoA and has been shown to be a critical modulator of RhoA-mediated actin dynamics (Amano et al., 2000). To examine the role of p160ROCK in monocyte tail retraction, we plated primary monocytes on coverslips in the presence of 10 μM of Y-27632 and then stained for F-actin to visualize cell morphology. Cells treated with the p160ROCK inhibitor showed morphological abnormalities similar to those observed in C3-loaded monocytes, albeit less severe (Fig. 6 A). The percentage of cells with tails was quantitated over a range of Y-27632 concentrations. We found that even at 1 μM, the percentage of cells with detectable tails nearly doubled over controls. Both the percentage of cells with tails, and the length of the unretracted tails increased with the concentration of Y-27632 (Fig. 6 A).

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