Leukocytes can rapidly migrate virtually within any substrate found in the body at speeds up to 100 times faster than mesenchymal cells that remain firmly attached to a substrate even when migrating. To understand the flexible migration strategy utilized by leukocytes, we experimentally investigated the three-dimensional modulation of cortical plasticity during the formation of pseudopodial protrusions by mouse leukocytes isolated from blood. The surfaces of viable leukocytes were discretely labeled with fluorescent beads that were covalently conjugated with concanavalin A receptors. The movements of these fluorescent beads were different at the rear, central, and front surfaces. The beads initially present on the rear and central dorsal surfaces of the cell body flowed linearly toward the rear peripheral surface concomitant with a significant collapse of the cell body in the dorsal-ventral direction. In contrast, those beads initially on the front surface moved into a newly formed pseudopodium and exhibited rapid, random movements within this pseudopodium. Bead movements at the front surface were hypothesized to have resulted from rupture of the actin cytoskeleton and detachment of the plasma membrane from the actin cytoskeletal cortex, which allowed leukocytes to migrate while being minimally constrained by a substrate. (C) 2013 Elsevier Inc. All rights reserved.