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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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Inhibition of RhoA causes a defect in tail retraction. (A) Primary monocytes were loaded with GST, C3, or RBD and plated on coverslips in serum-free media for 45 min before fixation and staining for F-actin. (B) Monocytes were loaded with GST, C3, or RBD and plated on coverslips in media containing 10% autologous serum for 45 min, followed by staining for F-actin (red) and tubulin (green). Bar, 20 μm.
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fig3: Inhibition of RhoA causes a defect in tail retraction. (A) Primary monocytes were loaded with GST, C3, or RBD and plated on coverslips in serum-free media for 45 min before fixation and staining for F-actin. (B) Monocytes were loaded with GST, C3, or RBD and plated on coverslips in media containing 10% autologous serum for 45 min, followed by staining for F-actin (red) and tubulin (green). Bar, 20 μm.

Mentions: We next explored the requirement for RhoA in monocyte spreading. First, we examined the actin cytoskeleton of cells loaded with either GST or C3, when plated on coverslips in serum-free medium. We observed profound morphological effects in cells treated with C3. The dramatic effect on monocyte morphology was observed as early as 15 min, and as late as 2 h after plating (data not shown). Although control cells adhered to the glass, but remained rounded, the C3-loaded cells spread and appeared to have long tails (Fig. 3 A). The F-actin in leukocytes accumulated in membrane ruffles and small punctate structures called podosomes (Marchisio et al., 1987). Actin polymerization at the leading edge is thought to drive forward movement of the cell, whereas podosomes contain many of the same proteins found in focal adhesions such as talin and vinculin. To further examine the organization of the cytoskeleton, we plated cells in the presence of serum to enhance spreading and migration. In addition, we assessed both the microtubule and actin structures. The microtubule network is an important cytoskeletal component that controls cell morphology, and its regulation is intimately tied to that of the actin cytoskeleton and Rho GTPases (Gundersen and Cook, 1999; Waterman-Storer et al., 1999; Ishizaki et al., 2001). The immunofluorescence micrographs in Fig. 3 B revealed that the core of the monocyte is filled with a microtubule network and that F-actin was prominent at the cell periphery. In sharp contrast, the C3-loaded cells were frequently polarized and appear to have long tails trailing behind the cell body. Note that there is a wide range of severity of this phenotype, but in all extended tails there is a significant microtubule network. In addition, we frequently observed prominent actin rich membrane protrusions in the tail.


RhoA is required for monocyte tail retraction during transendothelial migration.

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

Inhibition of RhoA causes a defect in tail retraction. (A) Primary monocytes were loaded with GST, C3, or RBD and plated on coverslips in serum-free media for 45 min before fixation and staining for F-actin. (B) Monocytes were loaded with GST, C3, or RBD and plated on coverslips in media containing 10% autologous serum for 45 min, followed by staining for F-actin (red) and tubulin (green). Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Inhibition of RhoA causes a defect in tail retraction. (A) Primary monocytes were loaded with GST, C3, or RBD and plated on coverslips in serum-free media for 45 min before fixation and staining for F-actin. (B) Monocytes were loaded with GST, C3, or RBD and plated on coverslips in media containing 10% autologous serum for 45 min, followed by staining for F-actin (red) and tubulin (green). Bar, 20 μm.
Mentions: We next explored the requirement for RhoA in monocyte spreading. First, we examined the actin cytoskeleton of cells loaded with either GST or C3, when plated on coverslips in serum-free medium. We observed profound morphological effects in cells treated with C3. The dramatic effect on monocyte morphology was observed as early as 15 min, and as late as 2 h after plating (data not shown). Although control cells adhered to the glass, but remained rounded, the C3-loaded cells spread and appeared to have long tails (Fig. 3 A). The F-actin in leukocytes accumulated in membrane ruffles and small punctate structures called podosomes (Marchisio et al., 1987). Actin polymerization at the leading edge is thought to drive forward movement of the cell, whereas podosomes contain many of the same proteins found in focal adhesions such as talin and vinculin. To further examine the organization of the cytoskeleton, we plated cells in the presence of serum to enhance spreading and migration. In addition, we assessed both the microtubule and actin structures. The microtubule network is an important cytoskeletal component that controls cell morphology, and its regulation is intimately tied to that of the actin cytoskeleton and Rho GTPases (Gundersen and Cook, 1999; Waterman-Storer et al., 1999; Ishizaki et al., 2001). The immunofluorescence micrographs in Fig. 3 B revealed that the core of the monocyte is filled with a microtubule network and that F-actin was prominent at the cell periphery. In sharp contrast, the C3-loaded cells were frequently polarized and appear to have long tails trailing behind the cell body. Note that there is a wide range of severity of this phenotype, but in all extended tails there is a significant microtubule network. In addition, we frequently observed prominent actin rich membrane protrusions in the tail.

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