Limits...
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.

Show MeSH

Related in: MedlinePlus

The contractility inhibitors, BDM and ML-7, do not induce tail formation. Monocytes were plated in serum containing media in the presence of various contractility inhibitors. After 45 min, cells were fixed and stained for F-actin to reveal cell morphology. (A) Control; (B) 20 mM BDM; (C) 20 μM ML-7; (D) 10 μM Y-27632. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196864&req=5

fig8: The contractility inhibitors, BDM and ML-7, do not induce tail formation. Monocytes were plated in serum containing media in the presence of various contractility inhibitors. After 45 min, cells were fixed and stained for F-actin to reveal cell morphology. (A) Control; (B) 20 mM BDM; (C) 20 μM ML-7; (D) 10 μM Y-27632. Bar, 20 μm.

Mentions: Both RhoA and its downstream effector, p160ROCK, are important regulators of actomyosin-based contractility (Chrzanowska-Wodnicka and Burridge, 1996; Kimura et al., 1996; Kureishi et al., 1997). In addition, myosin II is reported to be localized to the rear of migrating cells and the mechanical contractile force exerted by interactions between myosin II and actin are thought to be important for advancing the rear of the cell during migration (Lauffenburger and Horwitz, 1996; Sanchez-Madrid and del Pozo, 1999). Furthermore, it has been reported that blocking contractility with inhibitors of myosin light chain kinase (MLCK) results in unretracted uropods (Eddy et al., 2000). The uropod is a protrusion at the rear of polarized leukocytes that extends upwards from the cell body and contains high levels of various adhesion molecules which are important for mediating cell–cell interactions with other leukocytes (del Pozo et al., 1999; Sanchez-Madrid and del Pozo, 1999). To determine if inhibitors of actomyosin-based contractility could mimic the C3 phenotype in our monocytes, we treated cells with either 2,3-butanedione monoxime (BDM) or ML-7 and observed the effect on cell morphology using F-actin staining (Fig. 8) . BDM inhibits myosin ATPase activity, whereas ML-7 blocks the action of MLCK. BDM treatment did not induce tail formation, and instead the cells assumed a rounded morphology and were unable to spread. Due to the severe effect of BDM on monocyte spreading, we tried treating cells with BDM doses ranging from 0.1 to 20 mM, but failed to observe tails at any concentration (data not shown). To circumvent the spreading defect in BDM-treated monocytes, we added the inhibitor after cells had adhered and spread (15 min), but still observed rounded cells and no tails were formed (data not shown). Inhibition of MLCK by ML-7 was also not sufficient to inhibit tail retraction. At 20 μM ML-7, cells were indistinguishable from controls, and at 50 μM cells, became rounded (data not shown). In fibroblast control cells, both 20 mM BDM and 50 μM ML-7 were able to block actin stress fiber assembly (data not shown).


RhoA is required for monocyte tail retraction during transendothelial migration.

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

The contractility inhibitors, BDM and ML-7, do not induce tail formation. Monocytes were plated in serum containing media in the presence of various contractility inhibitors. After 45 min, cells were fixed and stained for F-actin to reveal cell morphology. (A) Control; (B) 20 mM BDM; (C) 20 μM ML-7; (D) 10 μM Y-27632. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: The contractility inhibitors, BDM and ML-7, do not induce tail formation. Monocytes were plated in serum containing media in the presence of various contractility inhibitors. After 45 min, cells were fixed and stained for F-actin to reveal cell morphology. (A) Control; (B) 20 mM BDM; (C) 20 μM ML-7; (D) 10 μM Y-27632. Bar, 20 μm.
Mentions: Both RhoA and its downstream effector, p160ROCK, are important regulators of actomyosin-based contractility (Chrzanowska-Wodnicka and Burridge, 1996; Kimura et al., 1996; Kureishi et al., 1997). In addition, myosin II is reported to be localized to the rear of migrating cells and the mechanical contractile force exerted by interactions between myosin II and actin are thought to be important for advancing the rear of the cell during migration (Lauffenburger and Horwitz, 1996; Sanchez-Madrid and del Pozo, 1999). Furthermore, it has been reported that blocking contractility with inhibitors of myosin light chain kinase (MLCK) results in unretracted uropods (Eddy et al., 2000). The uropod is a protrusion at the rear of polarized leukocytes that extends upwards from the cell body and contains high levels of various adhesion molecules which are important for mediating cell–cell interactions with other leukocytes (del Pozo et al., 1999; Sanchez-Madrid and del Pozo, 1999). To determine if inhibitors of actomyosin-based contractility could mimic the C3 phenotype in our monocytes, we treated cells with either 2,3-butanedione monoxime (BDM) or ML-7 and observed the effect on cell morphology using F-actin staining (Fig. 8) . BDM inhibits myosin ATPase activity, whereas ML-7 blocks the action of MLCK. BDM treatment did not induce tail formation, and instead the cells assumed a rounded morphology and were unable to spread. Due to the severe effect of BDM on monocyte spreading, we tried treating cells with BDM doses ranging from 0.1 to 20 mM, but failed to observe tails at any concentration (data not shown). To circumvent the spreading defect in BDM-treated monocytes, we added the inhibitor after cells had adhered and spread (15 min), but still observed rounded cells and no tails were formed (data not shown). Inhibition of MLCK by ML-7 was also not sufficient to inhibit tail retraction. At 20 μM ML-7, cells were indistinguishable from controls, and at 50 μM cells, became rounded (data not shown). In fibroblast control cells, both 20 mM BDM and 50 μM ML-7 were able to block actin stress fiber assembly (data not shown).

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