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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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Time-lapse analysis of monocyte movement. Monocytes loaded with either GST or C3 were cocultured with IL-1–activated endothelial monolayers, and video microscopy data were collected. Frames were taken every 20 s for 25 min. The sequences shown represent cell movement over 6.5 min. (A) GST-treated monocytes. (B) C3-loaded monocytes. (C) The movement of the x/y center of 10 GST- or C3-loaded monocytes was tracked for 15 frames. Each line represents the movement of a single monocyte over a 5-min period. (D) The ability of monocytes to invade the monolayer or to complete diapedesis was quantitated from analysis of video sequences from three separate experiments. Bars, 20 μm. Supplemental video is available at http://www.jcb.org/cgi/content/full/200103048/DC1.
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fig5: Time-lapse analysis of monocyte movement. Monocytes loaded with either GST or C3 were cocultured with IL-1–activated endothelial monolayers, and video microscopy data were collected. Frames were taken every 20 s for 25 min. The sequences shown represent cell movement over 6.5 min. (A) GST-treated monocytes. (B) C3-loaded monocytes. (C) The movement of the x/y center of 10 GST- or C3-loaded monocytes was tracked for 15 frames. Each line represents the movement of a single monocyte over a 5-min period. (D) The ability of monocytes to invade the monolayer or to complete diapedesis was quantitated from analysis of video sequences from three separate experiments. Bars, 20 μm. Supplemental video is available at http://www.jcb.org/cgi/content/full/200103048/DC1.

Mentions: Inhibition of RhoA by C3 has been described to cause membrane extensions that bear some resemblance to structures we observe in our monocytes (Allen et al., 1997; Kozma et al., 1997). To determine if the phenotype we observed in fixed cells was indeed a failure to retract the tail and not unregulated protrusions, we examined the movement of the monocytes on endothelial monolayers by video microscopy. Fig. 5 is a series of individual frames that show the movement of either GST-loaded (Fig. 5 A) or C3-loaded (Fig. 5 B) cells. The time elapsed between frames 1 and 6 is 6 min. The GST-loaded cell shown was migrating across the top of the endothelial monolayer, showing movement driven by lamellipodial extension at the leading edge, followed by retraction of the rear of the cell. For brief periods of time, control cells were observed to possess a short tail. In contrast, the sequence of C3-loaded cells shows a cell with a long tail and a main cell body that easily moves forward, but completely lacks tail retraction. Despite the forward crawling activity exhibited by the C3-loaded cell, the failure to retract the tail appears to draw the cell back to its starting point, eliminating any net translocation of the cell.


RhoA is required for monocyte tail retraction during transendothelial migration.

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

Time-lapse analysis of monocyte movement. Monocytes loaded with either GST or C3 were cocultured with IL-1–activated endothelial monolayers, and video microscopy data were collected. Frames were taken every 20 s for 25 min. The sequences shown represent cell movement over 6.5 min. (A) GST-treated monocytes. (B) C3-loaded monocytes. (C) The movement of the x/y center of 10 GST- or C3-loaded monocytes was tracked for 15 frames. Each line represents the movement of a single monocyte over a 5-min period. (D) The ability of monocytes to invade the monolayer or to complete diapedesis was quantitated from analysis of video sequences from three separate experiments. Bars, 20 μm. Supplemental video is available at http://www.jcb.org/cgi/content/full/200103048/DC1.
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Related In: Results  -  Collection

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

fig5: Time-lapse analysis of monocyte movement. Monocytes loaded with either GST or C3 were cocultured with IL-1–activated endothelial monolayers, and video microscopy data were collected. Frames were taken every 20 s for 25 min. The sequences shown represent cell movement over 6.5 min. (A) GST-treated monocytes. (B) C3-loaded monocytes. (C) The movement of the x/y center of 10 GST- or C3-loaded monocytes was tracked for 15 frames. Each line represents the movement of a single monocyte over a 5-min period. (D) The ability of monocytes to invade the monolayer or to complete diapedesis was quantitated from analysis of video sequences from three separate experiments. Bars, 20 μm. Supplemental video is available at http://www.jcb.org/cgi/content/full/200103048/DC1.
Mentions: Inhibition of RhoA by C3 has been described to cause membrane extensions that bear some resemblance to structures we observe in our monocytes (Allen et al., 1997; Kozma et al., 1997). To determine if the phenotype we observed in fixed cells was indeed a failure to retract the tail and not unregulated protrusions, we examined the movement of the monocytes on endothelial monolayers by video microscopy. Fig. 5 is a series of individual frames that show the movement of either GST-loaded (Fig. 5 A) or C3-loaded (Fig. 5 B) cells. The time elapsed between frames 1 and 6 is 6 min. The GST-loaded cell shown was migrating across the top of the endothelial monolayer, showing movement driven by lamellipodial extension at the leading edge, followed by retraction of the rear of the cell. For brief periods of time, control cells were observed to possess a short tail. In contrast, the sequence of C3-loaded cells shows a cell with a long tail and a main cell body that easily moves forward, but completely lacks tail retraction. Despite the forward crawling activity exhibited by the C3-loaded cell, the failure to retract the tail appears to draw the cell back to its starting point, eliminating any net translocation of the 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