SUMMARY Cell migration is a critical process for diverse (patho) physiological phenomena. Intriguingly, cell migration through physically confined spaces can persist even when typical hallmarks of 2D planar migration, such as actin polymerization and myosin II-mediated contractility, are inhibited. Here, we present an integrated experimental and theoretical approach (“Osmotic Engine Model”) and demonstrate that directed water permeation is a major mechanism of cell migration in confined microenvironments. Using microfluidic and imaging techniques along with mathematical modeling, we show that tumor cells confined in a narrow channel establish a polarized distribution of Na+/H+ pumps and aquaporins in the cell membrane, which creates a net inflow of water and ions at the cell leading edge and a net outflow of water and ions at the trailing edge, leading to net cell displacement. Collectively, this study presents an alternate mechanism of cell migration in confinement that depends on cell-volume regulation via water permeation.

Cell migration on planar surfaces is driven by cycles of actin protrusion, integrin-mediated adhesion, and myosin-mediated contraction; however, this mechanism may not accurately describe movement in 3-dimensional (3D) space. By subjecting cells to restrictive 3D environments, we demonstrate that physical confinement constitutes a biophysical stimulus that alters cell morphology and suppresses mesenchymal motility in human breast carcinoma (MDA-MB-231). Dorsoventral polarity, stress fibers, and focal adhesions are markedly attenuated by confinement. Inhibitors of myosin, Rho/ROCK, or β1-integrins do not impair migration through 3-μm-wide channels (confinement), even though these treatments repress motility in 50-μm-wide channels (unconfined migration) by ≥50%. Strikingly, confined migration persists even when F-actin is disrupted, but depends largely on microtubule (MT) dynamics. Interfering with MT polymerization/depolymerization causes confined cells to undergo frequent directional changes, thereby reducing the average net displacement by ≥80% relative to vehicle controls. Live-cell EB1-GFP imaging reveals that confinement redirects MT polymerization toward the leading edge, where MTs continuously impact during advancement of the cell front. These results demonstrate that physical confinement can induce cytoskeletal alterations that reduce the dependence of migrating cells on adhesion-contraction force coupling. This mechanism may explain why integrins can exhibit reduced or altered function during migration in 3D environments.

A vast amount of work has been dedicated to the effects of shear flow and cytokines on leukocyte transmigration. However, no studies have explored the effects of substrate stiffness on transmigration. Here, we investigated important aspects of endothelial cell contractionmediated neutrophil transmigration using an in vitro model of the vascular endothelium. We modeled blood vessels of varying mechanical properties using fibronectin-coated polyacrylamide gels of varying physiologic stiffness, plated with human umbilical vein endothelial cell (HUVEC) monolayers, which were activated with tumor necrosis factor-␣. Interestingly, neutrophil transmigration increased with increasing substrate stiffness below the endothelium. HUVEC intercellular adhesion molecule-1 expression, stiffness, cytoskeletal arrangement, morphology, and cell-substrate adhesion could not account for the dependence of transmigration on HUVEC substrate stiffness. We also explored the role of cell contraction and observed that large holes formed in endothelium on stiff substrates several minutes after neutrophil transmigration reached a maximum. Further, suppression of contraction through inhibition of myosin light chain kinase normalized the effects of substrate stiffness by reducing transmigration and eliminating hole formation in HUVECs on stiff substrates. These results provide strong evidence that neutrophil transmigration is regulated by myosin light chain kinase-mediated endothelial cell contraction and that this event depends on subendothelial cell matrix stiffness. (Blood. 2011;118(6):1632-1640) IntroductionLeukocyte transmigration through the vascular endothelium is a crucial step in the normal immune response. However, it is a complicated biologic process that involves many proteins and requires a coordinated effort between the leukocytes and endothelial cells (ECs). The biophysical aspects of leukocyte transmigration are also important, 1 as mechanical force transmission is an essential regulator of vascular homeostasis. It is probable that the mechanical properties of the vasculature depend on both vessel size (large vessels vs microvasculature) and location (soft brain vs stiffer muscle or tumor). Further, in the cardiovascular disease of atherosclerosis, the arteries stiffen 2-5 as an increased number of leukocytes penetrate the endothelium and tumor vasculature is also stiffer. 6 However, it is unknown how changes in vessel stiffness affect the behavior of the ECs lining the blood vessel, or the behavior of the leukocytes migrating along and transmigrating through the endothelium. Interestingly, polymorphonuclear neutrophils are capable of sensing differences in both substrate stiffness [7][8][9] and surface-bound adhesion proteins. 8 Therefore, we would expect neutrophils to be capable of sensing similar changes that may occur in their physiologic substrate, the endothelium.The mechanical properties of ECs are affected by a number of physiologic factors, including shear stress, 10 cholesterol content, 11,12 and oxidized low-density...

α4β1 integrin promotes migration of fibroblast-like cells in confined environment by enhancing myosin IIA via Rac1 inhibition, whereas unconfined migration requires Rac1 and myosin IIB.