Roles of Microtubule Dynamics and Small GTPase Rac in Endothelial Cell Migration and Lamellipodium Formation under Flow

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Abstract

Endothelial cell (EC) migration is required for vascular development and wound healing. We investigated the roles of microtubule (MT) dynamics and the small GTPase Rac in the fluid shear stress-induced protrusion of lamellipodia and enhancement of migration of bovine aortic ECs (BAECs). Shear stress increased lamellipodial protrusion and cell migration. Treating BAECs with paclitaxel (Taxol), an MT-stabilizing agent, inhibited lamellipodial protrusion and reduced migration speed in both the static and sheared groups. After Taxol washout, both lamellipodial protrusion and cell migration increased in the flow direction. Taxol treatment also decreased the shear-induced Rac activation. Transfection of BAECs with a dominant negative mutant of Rac1 inhibited lamellipodial protrusion and cell migration under static and shear conditions. Transfection with an activated mutant of Rac1 induced lamellipodia in all directions and attenuated the shear-induced migration, suggesting that an appropriate level of Rac activity and a polarized lamellipodial protrusion are important for cell migration under static and shear conditions. Our findings suggest that MT dynamics and optimum Rac activation are required for the polarized protrusion of lamellipodia that drives the directional EC migration under flow.

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Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness.

S. Palecek, J C Loftus, M Ginsberg … (1997)

Migration of cells in higher organisms is mediated by adhesion receptors, such as integrins, that link the cell to extracellular-matrix ligands, transmitting forces and signals necessary for locomotion. Whether cells will migrate or not on a given substratum, and also their speed, depends on several variables related to integrin-ligand interactions, including ligand levels, integrin levels, and integrin-ligand binding affinities. These and other factors affect the way molecular systems integrate to effect and regulate cell migration. Here we show that changes in cell migration speed resulting from three separate variables-substratum ligand level, cell integrin expression level, and integrin-ligand binding affinity-are all quantitatively predictable through the changes they cause in a single unifying parameter: short-term cell-substratum adhesion strength. This finding is consistent with predictions of a mathematical model for cell migration. The ligand concentration promoting maximum migration speed decreases reciprocally as integrin expression increases. Increases in integrin-ligand affinity similarly result in maximal migration at reciprocally lower ligand concentrations. The maximum speed attainable, however, remains unchanged as ligand concentration, integrin expression, or integrin-ligand affinity vary, suggesting that integrin coupling with intracellular motors remains unaltered.

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Microtubule growth activates Rac1 to promote lamellipodial protrusion in fibroblasts.

B. Liu, C Storer, K. Burridge … (1999)

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.

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Maximal migration of human smooth muscle cells on fibronectin and type IV collagen occurs at an intermediate attachment strength

JA Quinn, JA Stone, DA Lauffenburger … (1993)

Although a biphasic dependence of cell migration speed on cell- substratum adhesiveness has been predicted theoretically, experimental data directly demonstrating a relationship between these two phenomena have been lacking. To determine whether an optimal strength of cell- substratum adhesive interactions exists for cell migration, we measured quantitatively both the initial attachment strength and migration speed of human smooth muscle cells (HSMCs) on a range of surface concentrations of fibronectin (Fn) and type IV collagen (CnIV). Initial attachment strength was measured in order to characterize short time- scale cell-substratum interactions, which may be representative of dynamic interactions involved in cell migration. The critical fluid shear stress for cell detachment, determined in a radial-flow detachment assay, increased linearly with the surface concentrations of adsorbed Fn and CnIV. The detachment stress required for cells on Fn, 3.6 +/- 0.2 x 10(-3) mu dynes/absorbed molecule, was much greater than that on CnIV, 5.0 +/- 1.4 x 10(-5) mu dynes/absorbed molecule. Time- lapse videomicroscopy of individual cell movement paths showed that the migration behavior of HSMCs on these substrates varied with the absorbed concentration of each matrix protein, exhibiting biphasic dependence. Cell speed reached a maximum at intermediate concentrations of both proteins, with optimal concentrations for migration at 1 x 10(3) molecules/micron2 and 1 x 10(4) molecules/micron2 on Fn and CnIV, respectively. These optimal protein concentrations represent optimal initial attachment strengths corresponding to detachment shear stresses of 3.8 mu dyne/micron2 on Fn and 1.5 mu dyne/micron2 on CnIV. Thus, while the optimal absorbed protein concentrations for migration on Fn and CnIV differed by an order of magnitude, the optimal initial attachment strengths for migration on these two proteins were very similar. Further, the same minimum strength of initial attachment, corresponding to a detachment shear stress of approximately 1 mu dyne/micron2, was required for movement on either protein. These results suggest that initial cell-substratum attachment strength is a central variable governing cell migration speed, able to correlate observations of motility on substrata differing in adhesiveness. They also demonstrate that migration speed depends in biphasic manner on attachment strength, with maximal migration at an intermediate level of cell-substratum adhesiveness.