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.