ABSTRACT
Transendothelial migration of leukocytes involves the spatiotemporal regulation of adhesion molecules, chemokines and cytoskeletal regulators. Recent results show that distinct steps of leukocyte transendothelial migration are regulated by sequential integrin activation and coordinated Rho family GTPase activity. Progress has been made in understanding how the dynamic regulation of these molecules translates into leukocyte transmigration.
Subject(s)
Chemotaxis, Leukocyte , Endothelium, Vascular/immunology , Cell Adhesion Molecules/metabolism , Cell Movement , Chemokines/physiology , Cytoskeleton/physiology , Humans , Integrins/physiology , Models, Biological , rhoA GTP-Binding Protein/metabolismABSTRACT
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.
Subject(s)
Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Monocytes/metabolism , rhoA GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/physiology , Amides/pharmacology , CD18 Antigens/metabolism , Cell Adhesion , Cell Movement , Cells, Cultured , Chemokine CCL2/metabolism , Coculture Techniques , Cytoskeleton/metabolism , Dose-Response Relationship, Drug , Electroporation , Enzyme Inhibitors/pharmacology , Glutathione Transferase/metabolism , Humans , Interleukin-1/metabolism , Intracellular Signaling Peptides and Proteins , Microscopy, Fluorescence , Microscopy, Video , Microtubules/metabolism , Myosins/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Pyridines/pharmacology , Recombinant Fusion Proteins/metabolism , Time Factors , Umbilical Veins/cytology , rho-Associated KinasesABSTRACT
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.
