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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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C3-loaded monocytes mislocalize β2 integrin to the unretracted tail. Monocytes were loaded with GST or C3 and plated on coverslips in serum-containing media for 45 min before fixation and immunofluorescent localization of β2 integrin. The β2 integrin was concentrated at the leading edge of polarized GST control cells, whereas its expression was reduced in regions where the cell was retracting (arrows). In contrast, high levels of β2 integrin are visible in the unretracted tails of C3-loaded monocytes. Bars, 20 μm.
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fig10: C3-loaded monocytes mislocalize β2 integrin to the unretracted tail. Monocytes were loaded with GST or C3 and plated on coverslips in serum-containing media for 45 min before fixation and immunofluorescent localization of β2 integrin. The β2 integrin was concentrated at the leading edge of polarized GST control cells, whereas its expression was reduced in regions where the cell was retracting (arrows). In contrast, high levels of β2 integrin are visible in the unretracted tails of C3-loaded monocytes. Bars, 20 μm.

Mentions: Because we hypothesized that the effect of RhoA/p160ROCK on monocyte adhesion was localized to the rear of the migrating cells, we analyzed the distribution of β2 integrins in monocytes cultured on glass coverslips. Migrating monocytes assume a polarized morphology with a broad leading edge and more tapered tail region. Using this criteria, we indicated the rear of the monocytes with arrows in Fig. 10 . We found the β2 integrin localized at the leading edge of control monocytes. In contrast, we found high levels of β2 integrin in the unretracted tails of C3-loaded monocytes (Fig. 10). The ligand for β2 integrin in these samples is unclear, as the monocytes are plated on glass coverslips in the presence of serum. The β2 integrin is promiscuous and binds to charged surfaces, fibrinogen and fibronectin in addition to the more classical ICAM ligands (Rosen and Gordon, 1987; van den Berg et al., 2001; Yakubenko et al., 2001). Although these experiments showed mislocalization of β2 integrin on glass coverslips, the localization of the integrin in monocytes cocultured with endothelial cells remains to be determined. These results indicated that RhoA signaling was required for the appropriate localization of β2 integrin to the leading edge in monocytes cultured on coverslips. Together, the behavior of monocytes observed in the video microscopy, the adhesion assays, and the integrin localization data are consistent with a model in which RhoA and the p160ROCK signaling pathway are required to downregulate integrin-mediated adhesion in the rear of the migrating cell.


RhoA is required for monocyte tail retraction during transendothelial migration.

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

C3-loaded monocytes mislocalize β2 integrin to the unretracted tail. Monocytes were loaded with GST or C3 and plated on coverslips in serum-containing media for 45 min before fixation and immunofluorescent localization of β2 integrin. The β2 integrin was concentrated at the leading edge of polarized GST control cells, whereas its expression was reduced in regions where the cell was retracting (arrows). In contrast, high levels of β2 integrin are visible in the unretracted tails of C3-loaded monocytes. Bars, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig10: C3-loaded monocytes mislocalize β2 integrin to the unretracted tail. Monocytes were loaded with GST or C3 and plated on coverslips in serum-containing media for 45 min before fixation and immunofluorescent localization of β2 integrin. The β2 integrin was concentrated at the leading edge of polarized GST control cells, whereas its expression was reduced in regions where the cell was retracting (arrows). In contrast, high levels of β2 integrin are visible in the unretracted tails of C3-loaded monocytes. Bars, 20 μm.
Mentions: Because we hypothesized that the effect of RhoA/p160ROCK on monocyte adhesion was localized to the rear of the migrating cells, we analyzed the distribution of β2 integrins in monocytes cultured on glass coverslips. Migrating monocytes assume a polarized morphology with a broad leading edge and more tapered tail region. Using this criteria, we indicated the rear of the monocytes with arrows in Fig. 10 . We found the β2 integrin localized at the leading edge of control monocytes. In contrast, we found high levels of β2 integrin in the unretracted tails of C3-loaded monocytes (Fig. 10). The ligand for β2 integrin in these samples is unclear, as the monocytes are plated on glass coverslips in the presence of serum. The β2 integrin is promiscuous and binds to charged surfaces, fibrinogen and fibronectin in addition to the more classical ICAM ligands (Rosen and Gordon, 1987; van den Berg et al., 2001; Yakubenko et al., 2001). Although these experiments showed mislocalization of β2 integrin on glass coverslips, the localization of the integrin in monocytes cocultured with endothelial cells remains to be determined. These results indicated that RhoA signaling was required for the appropriate localization of β2 integrin to the leading edge in monocytes cultured on coverslips. Together, the behavior of monocytes observed in the video microscopy, the adhesion assays, and the integrin localization data are consistent with a model in which RhoA and the p160ROCK signaling pathway are required to downregulate integrin-mediated adhesion in the rear of the migrating cell.

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