Human neutrophil surface protrusion under a point load: location independence and viscoelasticity

Abstract

Mechanical properties of neutrophils have been recognized as key contributors to stabilizing neutrophil rolling on the endothelium during the inflammatory response. In particular, accumulating evidence suggests that surface protrusion and tether extraction from neutrophils facilitate stable rolling by relieving the disruptive forces on adhesive bonds. Using a customized optical trap setup, we applied piconewton-level pulling forces on targeted receptors that were located either on the microvillus tip (CD162) or intermicrovillus surface of neutrophils (CD18 and CD44). Under a constant force-loading rate, there always occurred an initial tent-like surface protrusion that was terminated either by rupture of the adhesion or by a "yield" or "crossover" to tether extraction. The corresponding protrusional stiffness of neutrophils was found to be between 0.06 and 0.11 pN/nm, depending on the force-loading rate and the cytoskeletal integrity, but not on the force location, the medium osmolality, nor the temperature increase from 22°C to 37°C. More importantly, we found that neutrophil surface protrusion was accompanied by force relaxation and hysteresis. In addition, the crossover force did not change much in the range of force-loading rates studied, and the protrusional stiffness of lymphocytes was similar to that of neutrophils. These results show that neutrophil surface protrusion is essentially viscoelastic, with a protrusional stiffness that stems primarily from the actin cortex, and the crossover force is independent of the receptor-cytoskeleton interaction. leukocyte rolling; optical trap; cell adhesion; microvillus; micropipette aspiration TO FIGHT against invading bacteria during the inflammatory response, human neutrophils leave circulating blood and migrate to infected tissues. This is a well-coordinated multistep journey that starts with weak rolling adhesion along the endothelial lining of the blood vessel, followed by firm adhesion and diapedesis. Stable rolling is an indispensable step to expose neutrophils to locally expressed signaling molecules that eventually activate the integrin-mediated firm adhesion. Driven by shear stress, neutrophil rolling is a highly complex and dynamic process mediated primarily by selectins and their ligands, including L-selectin and P-selectin glycoprotein ligand-1 (PSGL-1 or CD162) on neutrophils (37). L-selectin and PSGL-1 are selectively concentrated on the tips of neutrophil microvilli (1,4,21,42), which are actin-dependent, tiny surface protrusions that disappear when neutrophils are treated with actin-sequestering drugs such as latrunculin A and cytochalasin D (16, 38). On the contrary, ␤ 2 -integrins (CD18), which mediate firm adhesion after rolling, are confined to the intermicrovillus region of the neutrophil surface (4). This special arrangement of adhesion molecules helps coordinate the initiation of rolling adhesion and the subsequent firm adhesion.The capability of selectins and their ligands in mediating neutrophil rolling under physiological shear stress reli...

Constitutively active ezrin increases membrane tension, slows migration, and impedes endothelial transmigration of lymphocytes in vivo in mice

“…There are no comparable studies of membrane tension in other hematopoietic cells, but measurements in neutrophils using different technology measured a "cross-over force" (which may be functionally similar to our measurement of PTF) of approximately 40 to 50 pN. 33 A marked increase in membrane tension was observed in the ezrin T567E transgenic lymphocytes. Representative single traces (Figure 2A) illustrate that T567E transgenic cells require a higher force for tether formation (PTF) and for tether maintenance at a constant length (SSTF).…”

Flexural Rigidity and Shear Stiffness of Flagella Estimated from Induced Bends and Counterbends

“…The tip compliance (ratio of displacement to force) of Chlamydomonas flagella was measured with an optical tweezers system (29). Briefly, an 1)).…”

Membrane tubulovesicular extensions (cytonemes)

“…Membrane tethers can be extracted from the human neutrophils in vitro through micropipette manipulation or using laser tweezers. 5,6,9,12,21,22 The formation and shedding of membrane tethers or TVEs are involved in main aspects…”

“…The disruption of the actin cytoskeleton using latrunculin A or cytochalasin D relieved the pulling of membrane tethers from human neutrophils. 9,12,21,35 …”