Dynamics of Neutrophil Membrane Compliance and Microstructure probed with a Micropipet-based Piconewton Force Transducer

Abstract

A novel biointerface probe was implemented to study the deformability of the neutrophil membrane and cortical cytoskeleton. Piconewton scale forces are applied to the cell using an ultrasensitive and tunable force transducer comprised of an avidin-coated microsphere attached to a biotinylated and swollen red blood cell. Deformations of freshly isolated human neutrophils were observed on the stage of an inverted phase contrast microscope. Force versus probe indentation curves over a cycle of contact, indentation, and retraction revealed three distinct material responses. Small probe deformations (approximately 500 nm) tested over a range of rates (e.g. 100-500 nm/s) revealed predominantly an elastic response. An initial low-slope region in the force-indentation curves (approximately 0.005 pN/nm), typically extending 0.5-1.0 microm from the cell surface was interpreted as probe contact with microvilli extensions. Further deformation yielded a slope of 0.054+/-0.006 pN/nm, indicative of a stiffer cortical membrane. Disrupting cytoskeletal actin organization by pretreatment with cytochalasin D, reduced the slope by 40% to 0.033+/-0.007 pN/nm and introduced hysteresis in the recovery phase. Modeling the neutrophil as a liquid drop with constant surface tension yielded values of cortical tension of 0.035 pN/nm for resting and 0.02 pN/nm for cytochalasin-treated neutrophils. These data demonstrate the utility of the biointerface probe for measuring local surface compliance and microstructure of living cells.

A Neutrophil Timer Coordinates Immune Defense and Vascular Protection

“…However, binding analyses using soluble P-and E-selectin antibody chimeras revealed only modest changes in selectin binding, regardless of time of day or genetic background (Figure S4G), suggesting that biosynthesis of selectin ligands was unlikely to cause the loss of rolling during aging. Effective engagement of selectins under flow additionally demands correct topology at the neutrophil's surface to optimize ligand presentation at the tip of microvilli, a type of membrane protrusion that relies on a network of cortical actin (Finger et al, 1996;Simon et al, 2007;von Andrian et al, 1995). Analyses of actin distribution with immunofluorescence staining and of surface topology with scanning electron microscopy revealed dramatic reductions in cortical b-actin in aged neutrophils, which coincided with a reduced number of microvilli both in wild-type mice at ZT5 (Figures 4C and 4D) and in genetically induced aged mice (Cxcr4 DN ; Figures S4H and S4I).…”

Cytoskeletal prestress: The cellular hallmark in mechanobiology and mechanomedicine

“…Neutrophil deformation is measured after the cell is sucked into a micropipette (Evans & Kukan, 1984 ). Later a sensitive micropipette‐based piconewton (pN) (1 pN = 10 −12 Newton) force transducer to quantify neutrophil membrane stiffness dynamics has been developed (Simon et al, 2007 ). Adhering to TNFα (tumor necrosis factor‐alpha)‐activated endothelial cells, neutrophils elevate their stiffness within 2 min (Wang et al, 2001 ).…”

Mechanosensing at the Vascular Interface

“…Leukocytes possess microvilli with lengths of 300 to 700 nm. Upon cell contact and rolling on inflamed ECs, microvilli may provide a means of penetration through the GCX (97). Thus, interaction between receptors on ECs and their selectin ligands on rolling leukocytes would involve the penetration of microvilli through the GCX to form point contacts.…”