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 β2 integrin in the unretracted tails.
Microtubules are involved in actin-based protrusion at the leading-edge lamellipodia of migrating fibroblasts. Here we show that the growth of microtubules induced in fibroblasts by removal of the microtubule destabilizer nocodazole activates Rac1 GTPase, leading to the polymerization of actin in lamellipodial protrusions. Lamellipodial protrusions are also activated by the rapid growth of a disorganized array of very short microtubules induced by the microtubule-stabilizing drug taxol. Thus, neither microtubule shortening nor long-range microtubule-based intracellular transport is required for activating protrusion. We suggest that the growth phase of microtubule dynamic instability at leading-edge lamellipodia locally activates Rac1 to drive actin polymerization and lamellipodial protrusion required for cell migration.
Previously, we and others have shown that RhoA and ROCK signaling are required for negatively regulating integrin-mediated adhesion and for tail retraction of migrating leukocytes. This study continues our investigation into the molecular mechanisms underlying RhoA/ROCK-regulated integrin adhesion. We show that inhibition of ROCK up-regulates integrin-mediated adhesion, which is accompanied by both increased phosphotyrosine signaling through Pyk-2 and paxillin and inappropriate membrane protrusions. We provide evidence that inhibition of ROCK induces integrin adhesion by promoting remodeling of the actin cytoskeleton. Furthermore, we find that ROCK regulates membrane activity through a pathway involving cofilin. Inhibition of RhoA signaling allows the formation of multiple competing lamellipodia that disrupt productive migration of monocytes. Together, our results show that RhoA/ ROCK signaling promotes migration by restricting integrin activity and membrane protrusions to the leading edge.Circulating leukocytes respond to infection or injury by dramatically altering cell shape and adhesive properties to facilitate migration from the bloodstream to the affected tissue (1, 2). Leukocytes follow biochemical cues to guide the timing and location of activation (3, 4). These cues include soluble inflammatory cytokines and chemokines as well as stationary adhesion molecules. Both types of signals initiate immune system responses from resting leukocytes and provide directional information for appropriate migration.The process of cell migration can be described as the result of coordination between membrane protrusive activity at a leading edge, movement of the cell body, and retraction of the rear of the cell (5, 6). New membrane extensions at the leading edge are stabilized by adhesive interactions between integrins and stationary adhesion molecules located on another cell or deposited as an extracellular matrix. Conversely, the retraction of membrane at the rear of the cell requires the disruption of the adhesive interaction either by disengagement between the ligand and receptor or dissociation of the links between the integrin and the internal cytoskeleton (7).The actin cytoskeleton plays an important role in regulating both cell shape and adhesive properties. Actin polymerization at the leading edge can physically drive membrane protrusion (8). Interactions between the actin cytoskeleton and integrins regulate integrin activity (9, 10). The links between the actin cytoskeleton and integrins can either promote or restrain integrin adhesiveness depending on the cell type and environmental context. Early investigations of integrin-actin linkages in stationary fibroblasts demonstrated that actomyosin-dependent integrin clustering was required for strong integrin adhesions (11). More recently, a study of highly motile leukocytes and lymphocytes have revealed that when these cells are circulating, interactions between integrins and cortical actin can restrain integrin activity until the appropriate signals are received ...
ErbB-2/HER2 is an important signaling partner for the epidermal growth factor receptor (EGFR). Overexpression of erbB-2 is also associated with poor prognosis in breast cancer. To investigate how erbB-2 amplification affects its interactions with the EGFR, we used a human mammary epithelial cell system in which erbB-2 expression was increased 7-20-fold by gene transfection. We found that amplification of erbB-2 caused constitutive activation of erbB-2 as well as ligand-independent activation of the EGFR. Overexpression of erbB-2 strongly inhibited erbB-2 down-regulation following transactivation by EGFR. Significantly, down-regulation of activated EGFR was also inhibited by erbB-2 amplification, resulting in enhanced ligand-dependent activation of the EGFR. The rate of EGFR endocytosis was not affected by erbB-2 overexpression, but the rate of lysosomal targeting was significantly reduced. In addition, erbB-2 overexpression promoted rapid recycling of activated EGFR back to the cell surface and decreased ligand dissociation from the EGFR. Our data suggest that overexpression of erbB-2 inhibits both its downregulation and that of the EGFR. The net effect is increased signaling through the EGFR system.
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